Automated tissue engineering system

ABSTRACT

The invention provides systems, modules, bioreactor and methods for the automated culture, proliferation, differentiation, production and maintenance of tissue engineered products. In one aspect is an automated tissue engineering system comprising a housing, at least one bioreactor supported by the housing, the bioreactor facilitating physiological cellular functions and/or the generation of one or more tissue constructs from cell and/or tissue sources. A fluid containment system is supported by the housing and is in fluid communication with the bioreactor. One or more sensors are associated with one or more of the housing, bioreactor or fluid containment system for monitoring parameters related to the physiological cellular functions and/or generation of tissue constructs; and a microprocessor linked to one or more of the sensors. The systems, methods and products of the invention find use in various clinical and laboratory settings.

FIELD OF THE INVENTION

This invention relates to devices, methods and systems for the automatedculture, proliferation, differentiation, production and maintenance oftissue engineered products. Such systems, methods and products find usein various clinical and laboratory settings.

BACKGROUND OF THE INVENTION

Throughout this application, various references are cited in parenthesesto describe more fully the state of the art to which this inventionpertains. The disclosures of these references are hereby incorporated byreference into the present disclosure.

During the past several years, researchers have developed and useddifferent cell culture and tissue engineering techniques for the cultureand production of various types of cellular implants. Such systems aredescribed for example in U.S. Pat. Nos. 5,041,138, 5,842,477, 5,882,929,5,891,455, 5,902,741, 5,994,129, 6,048,721 and 6,228,635. Bioreactorsystems have also been developed for the culture of cells and cellularimplants and are described for example in U.S. Pat. Nos. 5,688,687,5,728,581, 5,827,729 and 6,121,042.

The aforementioned methods and systems generally employ conventionallaboratory culturing techniques using standard culture equipment forcell seeding of selected cell populations onto scaffolds. As such, thegenerated implants simply comprise proliferated cell populations grownon a type of biopolymer support where any manipulation of the cellularenvironment is limited to endogenous cell production of cytokinespresent in any standard cell culture, and application of shear and/orphysical stresses due to circulation of cell culture media and physicalmanipulation of the support onto which the cells are seeded. The systemsdo not address nor are they capable of generating a tissue implant thatcomprises proliferated and differentiated cells representative ofdeveloping tissues in vivo and further integrated within a selectedscaffold that can be successfully integrated in vivo. Moreover, knownmethods and systems are not capable of multi-functionally carrying outall of the steps of biopsy tissue digestion to yield disassociatedcells, subsequent cell seeding on a proliferation substrate, cell numberexpansion, controlled differentiation, tissue formation and productionof a tissue implant within a single automated tissue engineering system.This is primarily because known culture systems are not sophisticated inthat they are not capable of automatically evaluating and manipulatingthe changing environment surrounding the developing implant such thatcells progressively proliferate and differentiate into a desiredimplant.

Furthermore, conventional culture methods and systems are laborintensive and suffer from the drawbacks of contamination and varyingdegrees of culturing success due to human error and lack of continualperformance evaluation. Conventional culture systems require that mostof the initial steps in the preparation of cells for seeding (i.e.tissue digestion, cell selection) is performed manually which is timeconsuming, unreliable in terms of the quality of the tissue produced,and prone to culture contamination problems. The systems are incapableof supporting the automated preparation of tissue engineered implantsfrom primary or precursor cells due to inherent design limitations thatrestrict the cell and tissue culture process, the inability toadequately monitor and modify the environment to support tissuedevelopment, and the absence of techniques to enable the implementationof effective quality control measures.

Thus, there remains a real and unmet need for an improved system for invitro and ex vivo tissue engineering that can consistently meet theoperational requirements associated with the different steps in thedevelopment and production of tissue engineered implants. Of particularimportance is the ability to create functional tissue constructs wherethe cells present are active, differentiated and already expressingextracellular matrix. This involves more than, and is strikinglydifferent to, the simple simulation of the mature in vivo environmentpresent at the host site. This is because the preparation of functionalde novo tissue fundamentally requires that the cells progress through aseries of developmental stages as part of an ex vivo sequence.

In order to address both clinical and research requirements, newdevices, methods and systems have been developed that obviate several ofthe disadvantages and limitations of conventional ex vivo culturingtechniques and systems.

SUMMARY OF THE INVENTION

The present invention is directed to a user-friendly automated systemfor cell culture and tissue engineering that can be used in a variety ofclinical and research settings for both human and veterinaryapplications.

As used herein, “tissue engineering” may be defined as “the applicationof principles and methods of engineering and life sciences towardfundamental understanding and development of biological substitutes torestore, maintain and improve tissue functions”. This definition isintended to include procedures where the biological substitutes arecells or combinations of different cells that may be implanted on asubstrate or scaffold formed of biocompatible materials to form atissue, in particular an implantable tissue construct. Furthermore, itis noted that the cells involved in the tissue engineering processes maybe autologous, allogenic or xenogenic.

The tissue engineering system of the present invention is designed toperform all activities under sterile operating conditions. The system isfully automated, portable, multifunctional in operation andperforms/provides one or more of the following:

sterile reception/storage of tissue biopsy;

automated monitoring of digestion process

digestion of biopsy tissue to yield disassociated cells;

cell sorting and selection, including safe waste collection;

cell seeding on or within a proliferation substrate or scaffold

proliferation of cells to expand cell populations;

cell washing and cell collection;

cell seeding on or within a tissue engineering scaffold or matrix;

cell differentiation to allow specialization of cellular activity;

tissue formation;

mechanical and/or biochemical stimulation to promote tissue maturity;

harvesting the tissue engineered constructs/implants for reconstructivesurgery; and

storage and transportation of implantable tissue.

The tissue engineering system of the present invention may bepre-programmed to perform each of the above noted steps, individually,sequentially or in certain predetermined partial sequences as desiredand required. Furthermore, each of these steps, or any combinationthereof, are accomplished within one or more bioreactors on a tissueengineering module. In operation, the tissue engineering system ispre-programmed and automatically controlled thus requiring minimal userintervention and, as a result, enhances the efficiency andreproducibility of the cell culture and/or tissue engineering processwhile minimizing the risks of contamination. The tissue engineeringsystem of the invention and components thereof are operable underconditions of microgravity and/or zero gravity where such system andcomponents are used for space research.

The system of the present invention is designed such that primary orprecursor cells can be isolated from a donor tissue for furtherpropagation, differentiation and production of a tissue implant.Alternatively, cell lines may also be used either alone or incombination with other cell sources.

In accordance with the invention, is an automated tissue engineeringsystem, the system comprising a housing that supports at least onebioreactor that facilitates physiological cellular functions and thegeneration of tissue constructs from cell and tissue sources. Thehousing also supports a fluid containment system that is in fluidcommunication with the bioreactor. Associated with the housing and/orthe bioreactor are sensors that monitor physiological parameters offluid provided in the fluid containment system. A microprocessordisposed within the housing is linked to the bioreactor and the fluidcontainment system and functions to control their functioning. Themicroprocessor may also independently control environmental conditionswithin the system.

In accordance with another aspect of the invention there is provided asystem for cell and tissue engineering comprising portable, steriletissue engineering modules having one or more bioreactors which providethe basis for tissue digestion, cell seeding on a proliferationsubstrate, cell proliferation, cell seeding on a differentiationscaffold, cell differentiation, and tissue formation with subsequentmaturation into functional tissue for implantation. The bioreactor isoperatively connected with a media flow and reservoir system for thedelivery of reagents and the collection of waste fluids in a non-refluxmanner. The bioreactor and/or the media flow system optionally includegas exchange components that utilize semi-permeable membranes to allowthe transfer of gaseous products thereby controlling levels of dissolvedgases in the media. The tissue engineering module operatively interactswith a central microprocessor controlled base unit that automaticallymonitors the progression of the cell culture or tissue engineeringprocess and adjusts the environmental conditions to meet therequirements of the different stages of cell culture and tissuedevelopment within the bioreactor. Deviations from ideal conditions aresensed by a variety of sensors present within the bioreactor and thesignals generated are monitored by the central microprocessor. As such,changes in environmental conditions such as but not limited to pH,temperature and dissolved gases can be automatically monitored andaltered as required. In addition, the status of cell proliferation isindirectly assessed by detection of metabolic turnover as a function oftime (e.g. pH, O₂, CO₂, lactic acid and glucose consumption). Further tothe control of processing conditions by the central microprocessor, thetissue engineering module itself may optionally include a secondaryonboard microprocessor that operates in unison with the centralmicroprocessor. The tissue engineering module microprocessor expands thedata processing capabilities of the tissue engineering system byperforming specific functions directly onboard the tissue engineeringmodule, thereby minimizing the demands on the central microprocessor.

Various growth factors, cytokines, experimental agents, pharmaceuticals,chemicals, culture fluids and any combinations thereof may be loaded andstored within any of the reservoirs located on the tissue engineeringmodule and automatically transferred to the one or more bioreactorsaccording to a pre-programmed sequence or as required by the developingtissue implant. The individual tissue engineering modules are removablefrom the system for transport without compromising the sterility of thetissue engineered constructs present within the bioreactor. Such removaldoes not affect the processing of any other modules present within thetissue engineering system. Furthermore, the tissue engineering modulemay be considered to be disposable following the completion of a tissueengineering sequence, as this practice prevents contamination arisingfrom prior use.

In various embodiments of the invention, the device and system can beused to digest tissues obtained by surgical biopsy. In anotherembodiment, cells can be filtered and a particular population selectedand isolated. In another embodiment, digested cells can be proliferatedto expand the population of the cells. In still a further embodiment,cells can be seeded and cultivated on a desired scaffold or substrate(also referred to as a matrix). In yet a further embodiment, cells canbe differentiated on and/or throughout a desired scaffold or substrateuntil suitable tissue formation is obtained. In yet a furtherembodiment, the tissue may be stimulated to promote tissue maturity. Inyet another embodiment, a tissue implant is produced that is suitablefor reconstructive surgery. In still a further embodiment, cell samplingcan be done at each stage of cellular proliferation and developmentalprogression in a sterile manner without adverse effects on the cultureitself. Each of the aforementioned embodiments can be done alone orsequentially as desired. Tracking of such processing events can beperformed by the central microprocessor and/or the module-basedmicroprocessor for incorporation into quality control records.

In one aspect, the tissue engineering system optionally uses a syntheticbiomaterial compound, Skelite™, described in Applicant's U.S. Pat. No.6,323,146 (the contents of which are herein incorporated by reference)to enhance biological performance. Briefly, Skelite™ is an isolatedbioresorbable biomaterial compound comprising calcium, oxygen andphosphorous, wherein a portion of at least one of said elements issubstituted with an element having an ionic radius of approximately 0.1to 0.6 Angstroms. In one embodiment, Skelite™ may be used to enhancecell proliferation through its use as a coating on the walls of thebioreactor, as a thin film on the proliferation substrate, or as athree-dimensional and thereby high surface area proliferation scaffoldThe use of Skelite™ in the proliferation stage may be demonstrated to:

increase the rate of proliferation;

increase the cell yield following the proliferation step;

reduce the surface area required for a target cell yield;

reduce the problematic tendency of cell phenotype dedifferentiationduring proliferation; and

enhance the binding of growth factors to the proliferation substrate.

In a further embodiment, Skelite™ may be used as a resorbable scaffoldto enhance the differentiation of cells and the subsequent formation oftissue constructs. The use of Skelite™ in the differentiation stage maybe demonstrated to:

increase productivity by improving the reliability of thedifferentiation stage;

increase the integrity and hence biological viability of the tissueconstruct;

allow flexibility in construct configuration based on various scaffoldformats;

allow the stages of proliferation, differentiation and tissue formationto occur on a common substrate;

enhance the binding of growth factors to the differentiation scaffold;and

improve tissue construct handling properties during surgicalimplantation.

In another aspect, the present invention provides a method and systemfor the preparation of tissue constructs through the automated steps ofdigestion, proliferation, seeding and differentiation of primary orprecursor cells that originate from a patient thus eliminatingimmunological and disease transmission issues. An implant may be formedfrom the controlled cultivation of various cell types, including but notlimited to chondrocytes, stromal cells, osteoblasts, nerve cells,epithelial cells stem cells and mixtures thereof.

The system of the invention in an embodiment, incorporates one or moredetachable, portable, and independently operable tissue engineeringmodules that support one or more bioreactors, media reservoirs andfluid/media flow system. Each module, and hence the bioreactor(s), isunder the automated control of a central microprocessor. The module andassociated bioreactor(s) may be configured for various specializedapplications such as, but not limited to:

sterile reception/storage of tissue biopsy;

automated mixing and delivery of digestion reagents;

automated monitoring of digestion process;

digestion of biopsy tissue to yield disassociated cells;

cell sorting and selection, including safe waste collection;

cell washing and cell collection;

cell seeding on or within a proliferation substrate or scaffold;

automated mixing and delivery of proliferation reagents;

proliferation of cells to expand cell populations;

automated monitoring of cell conditions, including detection ofconfluence;

controlled cell release from the proliferation substrate or scaffold;

repeated proliferation steps on selected surface area sizes to increasecell numbers;

channeling of cell population toward one or more tissue engineeringscaffolds or matrices;

cell seeding on or within the tissue engineering scaffold or matrix;

automated mixing and delivery of differentiation reagents;

automatic monitoring of cell/tissue culture conditions;

cell differentiation to allow specialization of cellular activity;

tissue formation;

mechanical and/or biochemical stimulation to promote tissue maturity;

harvesting the tissue engineered constructs/implants for reconstructivesurgery; and

storage and transportation of cells and/or implantable tissue.

When two or more bioreactors, are provided within the system eithersupported directly within the housing of the system or supported on atissue engineering module insertable into the housing, the bioreactorsmay be provided connected in series and individually operable andcontrolled by the microprocessor or alternatively, may be operated andcontrolled independently depending on the user's programming of themicroprocessor and the desired result to be achieved. Furthermore, whentwo or more bioreactors are provided within the system, the bioreactorsand internal chambers may be connected such that there is an exchange ofcells and/or tissues from bioreactor to bioreactor.

The bioreactor can be manufactured in various sizes and configurationsas required to support varying numbers and sizes of proliferation anddifferentiation scaffolds or substrates. The bioreactor may beincorporated as part of the structural components of the tissueengineering module. Alternately, the bioreactor may be detachable as aseparate component to the remaining components of tissue engineeringmodule. If present as a discrete component, the bioreactor may bepackaged separately in a sterile package and joined to the tissueengineering module using sterile access techniques at the time of use.Furthermore, the sterile access techniques enable the bioreactor to bedetached from the module, upon completion of the tissue engineeringprocess, for easy transport to the operating room in preparation for theretrieval of a newly formed implantable tissue construct.

The bioreactor and/or the tissue engineering module may be rotated oragitated within the overall tissue engineering system via controlactuators. Rotation may enable the beneficial use of gravity to effectspecific bioprocessing sequences such as sedimentation-based cellseeding and fluid exchange within the bioreactor.

The tissue engineering module may be bar coded or provided with a memorychip for rapid and accurate tracking both within the tissue engineeringsystem and externally as part of the clinical or experimentalenvironment. Such tracking technology as incorporated within the tissueengineering device also enables electronic tracking via clinic-basedinformation systems for patient records. This ensures that the tissueengineering module and hence the associated cells or tissue implants areproperly coded to ensure administration to the correct patient and thatthe process is recorded for hospital billing purposes. The module and/orbioreactor may also utilize a bar code and/or memory chip in a similarmanner for rapid and accurate patient and sample tracking.

According to an aspect of the present invention is an automated tissueengineering system comprising;

a housing;

at least one bioreactor supported by said housing, said bioreactorfacilitating physiological cellular functions and/or the generation ofone or more tissue constructs from cell and/or tissue sources;

a fluid containment system supported by said housing and in fluidcommunication with said bioreactor,

one or more sensors associated with one or more of said housing,bioreactor or fluid containment system for monitoring parameters relatedto said physiological cellular functions and/or generation of tissueconstructs; and

a microprocessor linked to one or more of said sensors.

According to another aspect of the present invention is an automatedtissue engineering system comprising;

a housing;

at least one tissue engineering module removably accomodated within saidhousing, said tissue engineering module comprising a support structurethat holds at least one bioreactor, said bioreactor facilitatingphysiological cellular functions and/or the generation of one or moretissue constructs from cell and/or tissue sources, a fluid containmentsystem in fluid communication with said bioreactor, and one or moresensors for monitoring parameters related to said cell culture and/ortissue engineering functions; and

a microprocessor disposed in said housing and linked to said tissueengineering module, said microprocessor controlling the operation ofsaid tissue engineering module.

According to a further aspect of the invention is portable andsterilizable tissue engineering module, the module comprising;

a structural support holding at least one bioreactor, said bioreactorfacilitating cell culture and tissue engineering functions;

a fluid containment system in fluid communication with said bioreactor,and

one or more sensors for monitoring parameters related to said cellculture and tissue engineering functions.

In aspects of this embodiment, the bioreactor comprises a bioreactorhousing having one or more inlet ports and one or more outlet ports formedia flow and at least one chamber defined within said bioreactorhousing for receiving cells and/or tissues and facilitating said cellculture and tissue engineering functions. The chamber may be selectedfrom the group consisting of a cell culture/proliferation chamber, celldifferentiation/tissue formation chamber, tissue digestion chamber andcombinations thereof. Furthermore, the chamber houses one or moresubstrates and/or scaffolds. In embodiments of the invention, two ormore chambers may be provided operably connected within the bioreactorand be operably connected. Alternatively, the two or more bioreactorsmay be independently operable or co-operatively operable. In stillfurther aspects, the chambers and/or bioreactors are operably connectedto provide for the exchange of fluids, cells and/or tissues between thechambers and/or bioreactors. The scaffold for use in the presentinvention is selected from the group consisting of a porous scaffold, aporous scaffold with gradient porosity, a porous reticulate scaffold, afiberous scaffold, a membrane encircled scaffold and combinationsthereof. Chambers may also be further subdivided into zones. Forexample, a differentiation/tissue formation chamber may be provided witha plurality of zones to contain several scaffolds. Funnels or similarpassageways may be provided between chambers within a bioreactor.Furthermore, one or more filters may be provided at any location withina bioreactor.

According to still another aspect of the present invention is abioreactor that provides an environment for cell culture and/or tissueengineering functions selected from the group consisting of storage oftissue biopsy, digestion of tissue biopsy, cell sorting, cell washing,cell concentrating, cell seeding, cell proliferation, celldifferentiation, cell storage, cell transport, tissue formation, implantformation, storage of implantable tissue, transport of implantabletissue and combinations thereof.

According to still another aspect of the present invention is abioreactor for facilitating and supporting cellular functions andgeneration of implantable tissue constructs, said bioreactor comprising;

a bioreactor housing;

one or more inlet ports and one or more outlet ports for media flow;

at least one chamber defined within said bioreactor housing forfacilitating and supporting cellular functions and/or the generation ofone or more tissue constructs from cell and/or tissue sources; and

one or more sensors for monitoring parameters related to said cellularfunctions and/or generation of tissue constructs within said at leastone chamber.

In embodiments of the invention, the bioreactor housing comprises a lid,where the lid may be a detachable lid or integral with the bioreactorhousing.

Cells and tissues may be selected from bone, cartilage, related bone andcartilage precursor cells and combinations thereof. More specifically,cells suitable for use in the bioreactor, module and system of theinvention are selected from but not limited to the group consisting ofembryonic stem cells, adult stem cells, osteoblastic cells,pre-osteoblastic cells, chondrocytes, nucleus pulposus cells,pre-chondrocytes, skeletal progenitor cells derived from bone, bonemarrow or blood, including stem cells, and combinations thereof. Thecells or tissues may be of an autologous, allogenic, or xenogenic originrelative to the recipient of an implant formed by the cell culture andtissue engineering functions of the invention.

According to another aspect of the invention is a tissue implantproduced within a bioreactor of the present invention.

According to yet another aspect of the present invention is a tissueimplant produced by the tissue engineering system of the presentinvention.

According to another aspect of the present invention is a tissueengineered implantable construct for repair of bone trauma wherein theimplant comprises a porous scaffold of a bone biomaterial in combinationwith active bone cells and tissue engineered mineralized matrix.

According to another aspect of the present invention is a tissueengineered implant comprising:

a cartilage zone comprising tissue engineered cartilage that is devoidof any mineral-based scaffold;

a bone biomaterial zone comprising a porous scaffold; and

an interfacial zone between said cartilage zone and said bonebiomaterial zone.

The cartilage zone promotes lateral integration with the host cartilagewhile the bone biomaterial zone promotes lateral and verticalintegration with the subchondral bone plate when implanted in vivo. Theinterfacial zone provides the structural union between the cartilagezone and the bone biomaterial zone. The cartilage zone may additionallyincorporate a secondary non-mineral scaffold that assists with theformation of tissue engineered cartilage and allows for the developmentof a shaped surface profile in keeping with the particular anatomicalcharacteristics present at the site of implantation.

According to another aspect of the present invention is a method fordigesting a tissue biopsy, the method comprising;

loading a tissue biopsy within a bioreactor connected with a mediareservoir and flow system, said bioreactor having one or more sensors todetect physiological conditions within said bioreactor to amicroprocessor

providing tissue digestion enzymes; and

monitoring and maintaining suitable digestion conditions within saidbioreactor for a sufficient period of time for a desired level of tissuedigestion.

According to another aspect of the present invention is a method for theproliferation of cells, said method comprising;

seeding cells onto a proliferation substrate or scaffold supportedwithin a bioreactor connected with a media reservoir and flow system,said bioreactor having one or more sensors to detect physiologicalconditions within said bioreactor to a microprocessor; and

monitoring and maintaining suitable culturing conditions within saidbioreactor for a sufficient period of time for a desired level of cellproliferation.

According to another aspect of the present invention is a method for thedifferentiation of cells, said method comprising;

seeding cells onto a differentiation substrate or scaffold supportedwithin a bioreactor connected with a media reservoir and flow system,said bioreactor having one or more sensors to detect physiologicalconditions within said bioreactor to a microprocessor; and

monitoring and maintaining suitable culturing conditions within saidbioreactor for a sufficient period of time for a desired level of celldifferentiation.

According to another aspect of the present invention is a method fordigesting a tissue biopsy to provide primary cells, including precursorcells such as stem cells, and then proliferating and differentiating thecells to enable the formation of a tissue implant, the methodcomprising;

loading a tissue biopsy within a bioreactor connected with a mediareservoir and flow system, said bioreactor having one or more sensors todetect and relay physiological conditions within said bioreactor to amicroprocessor;

providing tissue digestion enzymes;

monitoring and maintaining suitable digestion conditions within saidbioreactor for a sufficient period of time to obtain disassociatedcells;

seeding the disassociated cells onto a proliferation substrate orscaffold supported within a bioreactor connected with a media reservoirand flow system, said bioreactor having one or more sensors to detectphysiological conditions within said bioreactor to a microprocessor;

monitoring and maintaining suitable culturing conditions within saidbioreactor for a sufficient period of time to obtain the desired levelof cell proliferation and expansion;

releasing the expanded cells from the proliferation substrate orscaffold;

seeding the expanded cells onto a differentiation substrate or scaffoldsupported within a bioreactor connected with a media reservoir and flowsystem, said bioreactor having one or more sensors to detect and relayphysiological conditions within said bioreactor to a microprocessor; and

monitoring and maintaining suitable culturing conditions within saidbioreactor for a sufficient period of time to obtain a tissue implant.

According to another aspect of the present invention is a method forproviding a skeletal implant, the method comprising;

seeding osteogenic and/or osteoprogenitor cells onto a porous scaffoldof a bone biomaterial supported within a bioreactor connected with amedia reservoir and flow system, said bioreactor having one or moresensors to detect physiological conditions within said bioreactor to amicroprocessor; and

monitoring and maintaining suitable conditions within said bioreactorfor a sufficient period of time to allow the osteogenic and/orosteoprogenitor cells to proliferate and/or differentiate throughout thescaffold to provide a tissue implant for orthopedic applications.

According to still another aspect of the invention is a method forproviding a cartilage implant, the method comprising;

seeding chondrogenic and/or chondroprogenitor cells onto a porousscaffold of a biomaterial supported within a bioreactor connected with amedia reservoir and flow system, said bioreactor having one or moresensors to detect physiological conditions within said bioreactor to amicroprocessor, and

monitoring and maintaining suitable conditions within said bioreactorfor a sufficient period of time to allow the chondrogenic and/orchondroprogenitor cells to proliferate and/or differentiate throughoutthe scaffold to provide a cartilage implant.

According to still another aspect of the invention is a method forwashing cells, the method comprising:

loading a cell suspension containing one or more undesired chemicalsinto a chamber;

continuously recirculating the cell suspension from the chamber througha cross-flow filtration module that comprises a membrane impermeable tosaid cells but permeable to said undesired chemicals to provide a washedcell suspension; and

collecting the washed cell suspension.

According to yet another aspect of the invention is a method forenrichment of cells, the method comprising:

loading a cell suspension containing excessive cell suspension volumeinto a chamber;

continuously recirculating the cell suspension from the chamber througha cross-flow filtration module that comprises a membrane impermeable tothe cells but allowing the excessive cell suspension volume to beremoved and collected.

According to yet another aspect of the invention is a method forproviding an implant for re-establishing the inner nucleus of a spinaldisc, the method comprising;

seeding nucleus pulposus cells within a scaffold a porous scaffold of abiomaterial supported within a bioreactor connected with a mediareservoir and flow system, said bioreactor having one or more sensors todetect physiological conditions within said bioreactor to amicroprocessor; and

monitoring and maintaining suitable conditions within said bioreactorfor a sufficient period of time to allow proliferation and/ordifferentiation of the nucleus pulposus cells and the expression ofextracellular matrix components characteristic of the nucleus pulposus.

According to still a further aspect of the present invention is a methodfor the preparation of quality assessment samples for use in clinicaltissue engineering, said method comprising;

parallel preparation of primary and secondary implants using the systemof the invention as described herein, where the primary implant is forimplantation and one or more secondary implants are for testing purposesto infer the calibre of the primary implant.

The tissue engineering system of the present invention in variousembodiments is under the control of one or more microprocessors that maybe preprogrammed in order that the user can select a specific type ofenvironment (or sequence of environments) within the bioreactor such astissue digestion, cell proliferation, cell differentiation and/or tissueconstruct formation. This eliminates operator intervention and reducesthe possibility of inadvertent contamination.

The tissue engineering system of the invention can be provided as a“kit”. In this manner the device, tissue engineering module(s),bioreactor(s) and various components thereof can be packaged and soldtogether along with instructions and quality control techniques.

The system of the present invention is ideal for clinical use inhospitals, and in particular surgical settings where due to traumaand/or disease, a tissue-engineered implant is desired. Using thepresent system, tissue engineered implantable constructs can be safelyprepared from autologous tissue obtained via patient biopsy, allogeniccells or xenogenic cells. The specifications of such tissue engineeredimplantable constructs can be matched to the type, size and condition ofthe implantation site. Furthermore, the implant as generated by thepresent system contains active cells that promote integration with thehost thereby improving patient recovery.

In practice, using an autologous cell model, a tissue biopsy can beobtained from the patient and placed directly into the bioreactorpresent on the tissue engineering module while in the operating room. Aspecific bioreactor design is selected depending on the type and size ofthe tissue construct desired. At the completion of the tissueengineering process, the tissue construct produced can be transportedstill contained in the sterile bioreactor to the operating room forimplantation back into the patient. The system is ideal for providing“customized” autologous tissue implants in a safe and therapeuticallyeffective manner.

The system and methods of the present invention are not limited toproviding automated cell culture techniques. The tissue engineeringsystem described moves well beyond the cell expansion used in celltherapy. The tissue engineering system may be used to create functionaltissue constructs where the cells present are active, differentiated andalready expressing extracellular matrix. Consequently, the tissueconstructs so produced are in a high state of development and therebyaccelerate the rate and improve the quality of tissue repair at theimplant site.

The system of the invention is also suitable for pharmacologicalresearch. Specifically, the system finds use in the area of drugdevelopment. New potential drugs and molecules can be tested on cellsand tissues to determine effects on cellular events and tissuedevelopment. Such testing can be done on a patient's own cells/tissuesto assess and possibly avoid adverse side effects prior toadministration. Alternatively, specialized cell lines or tissues can beused with the system as a key tool in the drug discovery process. Thesystem can be programmed to monitor and assess various physiologicalconditions of the cells/tissues present within the bioreactor and thusprovide a fast indication of the biological effects of a selected drugor molecule.

The system may also be used for research and development studies whereconventional tissue engineering techniques are difficult to use andpractice, and/or in conditions requiring extensive diagnostic recording.For example, microgravity studies involving tissue engineering aredifficult to conduct due to the unique properties of this environment.Traditional cell and tissue culture techniques are simply not viable inthis environment due to fluid containment issues and the absence ofgravity-based transport of cells. The system and methods of theinvention are easily adaptable to the microgravity environment as thesystem is completely sealed to prevent fluid loss and the migration ofcells as part of the tissue engineering process can be achieved bycontrolled fluid flow.

Other features and advantages of the present invention will becomeapparent from the following detailed description, examples and drawings.It should be understood, however, that the detailed description,specific examples and drawings while indicating embodiments of theinvention are given by way of illustration only, since various changesand modifications within the spirit and scope of the invention willbecome apparent to those skilled in the art from said detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be further understood from the followingdescription with reference to the figures, in which:

FIG. 1 illustrates a general methodology for clinical tissue engineeringas applied to the example of cartilage repair using autologouschondrocytes;

FIG. 2 shows an integrated tissue engineering device of the presentinvention;

FIG. 3 shows a further embodiment of the tissue engineering device ofFIG. 2;

FIG. 4 shows a further embodiment of the tissue engineering device ofFIG. 2;

FIG. 5 shows a cut-away view of the tissue engineering device of FIG. 2illustrating some of the internal components and a tissue engineeringmodule for insertion into the device;

FIG. 6 shows an enlarged cut-away view of the tissue engineering deviceof FIG. 2 illustrating an inserted tissue engineering module;

FIG. 7 shows an enlarged perspective view of the tissue engineeringmodule and interface with components of the device housing;

FIG. 7(a) shows an enlarged perspective view of the bioreactor and pumpunit;

FIG. 7(b) shows an enlarged perspective view of the pump unit and theassociated pump tubing;

FIG. 8 shows a perspective view of the reverse side of the tissueengineering module of FIG. 7 and the internal configuration of the flowplate that attaches thereto;

FIG. 9 shows an enlarged perspective view of the mixing andmicro-loading components associated with the instrumented bioreactordesign;

FIG. 10 shows the basic tissue engineering fluid flow schematic;

FIG. 11 shows a further embodiment of the basic tissue engineering fluidflow schematic;

FIG. 12 shows alternate bioreactor, proliferation substrate or scaffold,differentiation scaffold and process monitoring designs, as applicableto different tissue engineering scenarios;

FIG. 13 shows an enlarged perspective view of the bioreactor of thetissue engineering module, illustrating the internal configuration ofthe bioreactor and the flow path of fluids;

FIG. 14 shows a further embodiment of the bioreactor of the tissueengineering module, illustrating the internal configuration of thebioreactor;

FIG. 15 shows a rotatable bioreactor design;

FIG. 16 shows the sterile sampling embodiment of the tissue engineeringmodule;

FIG. 17 shows a further embodiment of the tissue engineering fluid flowschematic;

FIG. 18 shows yet a further embodiment of the tissue engineering fluidflow schematic; and

FIG. 19 shows a bioreactor design suitable for tissue digestion and cellcollection;

FIG. 20 shows a bioreactor design suitable for cell proliferation;

FIG. 21 shows a bioreactor design suitable for cell differentiation andtissue construct formation;

FIG. 22 shows yet a further embodiment of the tissue engineering fluidflow schematic; and

FIG. 23 shows a further embodiment of the tissue engineering module withseparate bioreactors for tissue digestion/cell collection, cellproliferation, and cell differentiation/tissue formation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to an integrated, automated tissueengineering device for the ex vivo processing of cells, particularlyautologous cells, to enable cell proliferation, cell differentiation andtissue formation in an efficient and consistent manner requiring minimalhuman intervention. The tissue constructs developed within the devicemay be integrated into a host to assist in tissue reconstructionprocedures and subsequent patient recovery. Furthermore, the inventionprovides automated methods for tissue engineering using a variety ofcells from a number of different sources (for example autologous cellsobtained via patient biopsy, allogenic cells or xenogenic cells).Furthermore, the cells may be precursor cells, primary cells, cells froman immortal cell line and combinations thereof.

The general methodology and principle for clinical tissue engineeringincorporating the tissue engineering system and methods of the presentinvention is illustrated in FIG. 1, using autologous cartilage tissueengineering as a representative example. In such example, cells (i.e.chondrocytes) are obtained from a surgical biopsy of a patient andeither manually or automatically seeded onto a suitable substrate orscaffold (i.e. a Skelite™ support). The chondrocytes and the support arepresent within the bioreactor portion of an automated tissue engineeringmodule, with the module forming part of a clinical base station of thetissue engineering system. A central microprocessor is present withinthe tissue engineering system and controls and customizes the internalenvironment of the bioreactor, and hence facilitates tissue growththerein, resulting in the stimulation of cell growth within and onto thesupport to generate an implant. Sensors within the bioreactor providefeedback to the microprocessor to ensure that the cells are seeded,expanded and differentiated in a desired and controlled manner toprovide an autologous tissue implant. Once the implant is generated, itis removed from the bioreactor for surgical implantation into thepatient. The present system provides an advantageous way to provideautologous tissue engineered implants in a sterile, safe, convenient andefficacious manner. Furthermore, the ability to prepare tissueengineered implants in a clinical setting allows considerableflexibility in the locations for undertaking the tissue engineeringprocess. While the system can be used in a centralized location, thedesign and operation of the system enables clinical use at regionalcenters. Such widespread availability precludes the transportation ofbiological material to and from centralized cell/tissue processingfacilities, thereby improving the cost effectiveness and efficiency ofthe tissue engineering process while avoiding shipment, tracking andregulatory complications.

In accordance with an embodiment of the present invention is a tissueengineering system as shown in FIG. 2 and generally indicated withreference numeral 100. The system 100 (may alternatively be referred toas a device) comprises a housing 102 having an insertion slot 104 forreceiving a tissue engineering module. The insertion slot 104 has amovable door 106 and a locking mechanism 108. A user interface 110 suchas a touch screen, key pad or combination of both is provided forcontrol of system operation and for the display of system status. A datastorage system 112 is present which permits the recording of informationvia a variety of mediums known to those of skill in the art (i.e. ZIP,CDROM, diskette, flashcard). A computer/communications link 114 providesthe capability to upload new software, modify control parameters usingan external computer, download data as well as troubleshoot and test thedevice. This link also permits the system to be connected to electronicinformation systems present at the clinic. The system 100 is poweredwith a power input 116. FIG. 3 shows a further embodiment of the system100 having several bay doors 106 to accommodate several tissueengineering modules. FIG. 4 shows a further embodiment of the system 100having bay doors 106 orientated in a horizontal manner to allow for thepreferential orientation of the tissue engineering module relative tothe gravity vector.

FIG. 5 shows the internal configuration of the system 100 represented inFIGS. 2 and 3 with the vertical orientation of the bay doors forvertical insertion of a tissue engineering module. A tissue engineeringmodule 118 is shown for insertion within the insertion slot 104 of thebay door 106. The tissue engineering module 118 slides into the systemhousing 102 via a guide rail system 120. Upon insertion, the module 118engages with one or more pump units 122 (i.e. peristaltic, piston,diaphragm or rotary), electrical connectors 124 (i.e. DIN, AMP, PCB,breadboard socket), and valve actuators 126 (i.e. servo motor, lineardrive, linear actuator). Any suitable guide system to allow the moduleto be inserted properly into the system may be contemplated as isunderstood by one of skill in the art.

As better seen in FIG. 6, where the tissue engineering module 118 isinserted into the housing 102, a series of valve actuators 126 interfacewith valves (shown in more detail in FIGS. 7 and 7 a) on the module toprovide flow control. The electrical connectors 124 provide electricalconnection between the module 118 and a central microprocessor unit(CPU) 128 via an electronic back-plane 130. The CPU 128 controls theoperational sequence, the transport of fluids and gases, the managementof process data, the monitoring of system status, the user interface,and the external data communication port. The CPU 128 provides controlthrough electrical links with active and passive electrical componentspresent on the back-plane 130 and each of the inserted tissueengineering modules 118.

Temperature sensors 132 (i.e. thermocouple, RTD or thermistor), gassensors 134 (i.e. O₂ and CO₂) and an environment control unit (ECU) 136are controlled by the CPU 128 to maintain the environment (i.e.temperature and gas atmosphere) within the housing 102 using standardmethods known to those skilled in the art. The environment can beadjusted to meet the requirements of the tissue engineering process,including storage of reagents at refrigeration temperature (i.e. 4° C.),the simulation of nominal body temperature (i.e. 37° C.), and theavailability of gaseous mixtures for transport into and out of themodule 118 in the event that the module is equipped with gas exchangecomponents (i.e. membranes). Gaseous conditions are monitored by the gassensors 134 located within the housing 102 and the data is sent to theCPU 128 via the electronic back-plane 130. Gas input(s) to the ECU canbe via gas supply inlet 140 provided within the housing 102 configuredwith standard fittings. In other embodiments, gases may be housed withinthe ECU. Gases for use within the device include but are not limited tooxygen, carbon dioxide, nitrogen and mixtures thereof. In order toadequately contain such gases within the housing 102, the bay door 106is configured to provide for a hermetic seal when closed. The housing102 is insulated with insulating material 142 such as styrofoam,aerogel, fiberglass and the like to allow for the efficient regulationof internal temperatures (i.e. 4° C. to 37° C.).

While the tissue engineering system of the present invention isgenerally shown to comprise a boxed shaped housing, it is understood byone of skill in the art that the housing may be made of variousconfigurations so long as it may accommodate the components as describedherein. For example, this includes but is not limited to openconfigurations that may not require a top and/or side portions.

The tissue engineering module 118 is illustrated in more detail in FIGS.7-9. The tissue engineering module 118 comprises a rigid structuralspine 200 to which is affixed a bioreactor 202. The bioreactor 202comprises a bioreactor housing that has a lid 204 and may be customizedwith respect to the substrate(s) or scaffold(s) contained therein toenable tissue digestion, cell culture, cell proliferation, celldifferentiation, tissue implant formation and combinations thereof. Thebioreactor lid may be detachable or alternatively made integral to thebioreactor housing. The bioreactor 202 may be separately detachable anddisposable relative to the structural spine 200. To enable suchdetachment, the bioreactor 202 and the structural spine 200 may usefluid disconnect fittings that include the provision for self sealing ofinput and output lines to avoid loss of fluids and to preventcontamination of the contents of the bioreactor. The entire tissueengineering module may be considered to be disposable following thecompletion of a tissue engineering sequence, as this practice preventscontamination arising from prior use. Alternately, only selectedcomponents of the module 118 may be considered as disposable due tocontact with fluids, leaving non-contamination prone componentsavailable for re-use.

As seen in FIGS. 7, 7 a and 7 b, a fluid containment system 206 isaffixed onto the structural spine 200 of the tissue engineering module118. The fluid containment system 206 is comprised of a sterile seriesof flexible reservoirs 208 and flexible tubing 210 for supplying andretrieving types of tissue and cell culture fluids and pharmaceuticalsto and from the bioreactor 202. The reservoirs 208 may be of varyingconfiguration and number as required and may contain different types ofcell and tissue culture media, growth factors, pharmaceutical agents andmay also contain waste media and/or media samples from the bioreactor202. Fluids are loaded or removed from the fluid containment system 206via a series of fluid access ports 212. Tubing 210 is present to providefluid connection between the various reservoirs 208 and the fluidcontrol components, such as the fluid flow control valves 214. The fluidflow control valves 214 are opened and closed by valve actuators 126.Similarly, the pump unit 122 interfaces with disposable pump componentspresent on the module. These pump components may be pistons, diaphragms,rotary elements or peristaltic tubing 218, provided that the operationof these components does not generate harsh conditions, such asexcessive shear stress, that compromise cell viability during thetransfer of cell suspensions. The pump unit 122 and the valve actuators126 reside within the housing 102. Alternately, the actuators and pumpunit may form part of the tissue engineering module, however, this mayresult in disposal of these components following patient use. Fluid istransferred out of the reservoirs 208 by the programmed action of thepump unit 122 on the pump tubing 218. Fluid travels from a flexiblefluid reservoir 208 to a fluid valve 214 via tubing 210. A fluid flowplate 220 (as shown in FIG. 8) directs fluid flow between different flowcontrol valves 214 and the pump tubing 218 of the pump unit 122. Fluidis returned to a selected empty reservoir 208 for storage. A flexibleprinted circuit board (PCB) 222 provides the electronic interface forelectronic components (i.e. sensors) present on the structural spine 200and/or the bioreactor 202. In the event that a sensor indicates that amonitored parameter (e.g., pH) is outside acceptable levels, the CPUtriggers a control intervention such as replacing the media within thebioreactor.

The tissue engineering module may optionally include a microprocessor224 to enable data processing and data storage directly on the module.This information may transferred to the central CPU 128 while the moduleis inserted into the housing 102 and retained in electronic memory forlater access once the module is removed. In addition to the data storedvia the microprocessor or memory chip resident on the tissue engineeringmodule, the module may also optionally include a bar code 226, magneticstrip 228, electronic memory (not shown) and/or ID label 230 tofacilitate administrative tracking within the clinic.

As seen in FIG. 8, the fluid flow plate 220 is secured to the 25structural spine 200 of the tissue engineering module 118. The techniquefor attachment of the fluid flow plate may be, but is not limited to, apress fit, snap fit, ultrasonic weld, solvent bond and the like,recognizing that the technique adopted must allow for sealing of theassembly to avoid loss of fluids and to prevent contamination. As shownin the disassembled view in FIG. 8 a, the fluid flow plate 220 has anintegral fluid pathway 232 to provide a means for directing flowassociated with the actuation of the fluid valves 214. New flow pathsmay be accommodated via revisions to the pathway present on the flowplate 220. In one embodiment, the fluid plate 220 may be integrallyformed into the structural spine 200 to form a single component. A fluidheating and mixing chamber 234 is included to ensure fluids that aredirected to the bioreactor are at the correct temperature and areadequately mixed so as to not disrupt the biological processes underwayin the bioreactor. Furthermore, a thermoelectric element 236 is presenton the tissue engineering module 118 to vary the temperature within thebioreactor 202 compared with the operational temperature of the module,as defined by the operation of the ECU 136. Such a temperature changemay be necessary to simulate nominal physiological conditions within thebioreactor, while the remaining components of the tissue engineeringmodule, particularly the reagents and/or samples, are at a reducedtemperature (i.e. refrigeration) to maintain physical, chemical and/orbiological viability. Power and control of the thermoelectric element isperformed by the CPU 128. In addition to sensors present on thebioreactor, a sensor 238 present on the tissue engineering moduleprovides feedback to the CPU 128. The sensor and thermoelectricconnections are made via the electrical cabling 240 and connector 124.

FIG. 9 shows the mixing and micro-loading aspects of the tissueengineering module 118. The bioreactor 202 has a mixing drive 260operably connected with a mixing actuator 262 and to the mixingdiaphragm 264. The mixing diaphragm is incorporated as part of thebioreactor 202 or the bioreactor lid 204, as shown. In operation, themixing drive 260 in combination with the mixing actuator 262 providetranslation or pulsing of the mixing diaphragm to effect controlledmixing of the contents of the bioreactor 202. Ideally, the nature of themixing is such to avoid high fluid shear that could compromise thephysical integrity of cells present within the bioreactor. For certaintissue engineering protocols, moderate levels of fluid shear areactually beneficial for the successful development of tissue constructs.In addition to the mixing components, an impact drive 266 and impactactuator 268 are present. These components serve to apply a controlledimpact to the bioreactor assembly at the conclusion of the proliferationsequence to assist with the release of cells from a proliferationsubstrate or scaffold resident within the bioreactor. Also provided is amicro-loading drive 270 in operable connection with a micro-loadingactuator 272 and micro-loading diaphragm 274. The micro-loadingdiaphragm 274 is incorporated as part of the bioreactor 202 or thebioreactor lid 204, as shown. The location and orientation of themicro-loading diaphragm is such to enable intimate contact with thesubstrate or scaffold and any associated cells or tissues present in thebioreactor 202. The application of micro-loads is known to beadvantageous for certain tissue engineering protocols. The mixing drive260, impact drive 266, and micro-loading drive 270 may be any of aseries of electromechanical devices such as solenoids, linear drives,rotational drives, or piezo electric components. Furthermore, it ispossible for the mixing drive 260, impact drive 266, micro-loading drive270, and the related actuators to be mounted on the housing 200.Alternatively, the drives and actuators may be mounted on the tissueengineering module or the bioreactor provided that the design of thedrives is consistent with the disposable nature of the tissueengineering module. In addition to the provision of mechanicalstimulation, the bioreactor may also be configured to introduceelectrical and/or chemical stimulation of the tissue construct. Inparticular, electric fields may be generated in the region of thebioreactor to enhance cell transport and/or tissue formation. Methods ofgeneration of electric fields are known to those of skill in the art andinclude but are not limited to the provision of electric coils.

FIG. 10 illustrates a basic fluid flow schematic for the tissueengineering module 118 in which there is a single cell or tissue culturechamber present within the bioreactor 202, (refer to descriptions ofFIGS. 12 and 17 for further information on the multi-chamberbioreactors). The flow path links the bioreactor 202 to reservoirs 208that supply fluid and collect waste. The fluid access ports 212 may beused to load reagents or remove samples or waste fluid. Flow isgenerated by the operation of pump unit 122 with flow direction definedby actuation of specific flow control valves 214. Perfusion to thebioreactor can be either continuous or pulsatile, provided that theassociated flow does not result in high fluid shear in regionscontaining cells, as such conditions could damage the cells or anemerging tissue construct. A recirculation loop 280 is provided to allowthe fluid contents of the bioreactor to be either monitored or modifiedby external components, such as an in-line gas exchange membrane 282,without necessitating the delivery of new fluid from the fluidreservoirs 208. Components of the tissue engineering module 118dedicated to storing fluids, (i.e. reservoirs 208), are keptrefrigerated at approximately 4° C. to facilitate storage of fluids thatwould otherwise degrade at the elevated temperatures used to simulatebody temperature (i.e. 37° C.). According to a preprogrammed routine,the CPU 128 controls the operation of fluid valve(s) 214 to allow fluidstored in a reservoir 208 to be delivered via the pump unit 122 into aheating and mixing chamber 234 prior to entry into the bioreactorchamber 300 (shown in detail in FIGS. 13, 14 and 15). Fluids aresupplied to the bioreactor via the inlet port 302 and removed via outletport 304. To simulate normal body temperatures for optimal cell andtissue culture performance, the bioreactor 202, the pump unit 122 andthe heating and mixing chamber 234 are maintained at approximately 37°C. by the operation of a thermoelectric element 236. It will be obviousto one skilled in the art that alternate thermal regulation devices maybe used to obtain the desired thermal profiles for the tissueengineering module 118.

FIG. 11 illustrates a variation on the basic fluid flow schematic wherethe fluid flow control valves are substituted for multiple pump units122. This configuration provides enhanced operational redundancy and areduced component count. Operation of such a system requires that anydormant pump unit prevents unregulated pass-through flow, as such anoccurrence would compromise the controlled delivery of fluids.

FIGS. 12 a-12 d illustrate various bioreactor configurations andalternate formats for the substrates and scaffolds used for theproliferation and differentiation steps involved in the operation of thetissue engineering system. FIG. 12 a shows a series of interchangeablebioreactor designs that address different bioprocessing scenarios. TheType I scenario is indicative of a basic single chamber 300 within abioreactor 202 that accommodates a proliferation scaffold or substrate310, or a differentiation scaffold or substrate (not shown) and isideally suited to either proliferation or differentiation. Cells areeither manually seeded onto the scaffold 310 or automatically deliveredvia the fluid pathway of the tissue engineering module.

The Type II scenario involves a multi-chamber bioreactor that providesfor the use of a scaffold 310 (or substrate) for proliferation of thecell population and an implantable differentiation scaffold 312 thatpromotes the formation of a tissue construct. The culture/proliferationchamber 300 is connected to the differentiation/tissue formation chamber306 via a funnel 314. The funnel serves to channel the cells releasedfrom the proliferation scaffold 310 into the implantable differentiationscaffold 312. The use of a filter 316 in several locations within thebioreactor serves to regulate the size of the cells or cell aggregatesthat can freely pass from one chamber to the next. A filter 316 a ispresent upstream of the proliferation scaffold with the purpose ofregulating the incoming cell population for the cell expansion step.Another filter 316 b is present upstream of the differentiation scaffoldagain to control the cell population entering this step of the tissueengineering sequence. In addition, there is a further filter 316 c overthe outlet port 304 to prevent the loss of cells from thedifferentiation/tissue formation chamber during operations involvingfluid transfer through the bioreactor. The filter 316 can be a filtermembrane or mesh or similar type filtering material as is known to thoseof skill in the art.

The Type III scenario combines tissue digestion with subsequentproliferation, differentiation and tissue construct formation. In thisscenario, a tissue biopsy 320 is loaded into a digestion chamber 322present within the bioreactor 202. Digestion of the tissue biopsy occursthrough the delivery of digestion enzymes into the bioreactor from oneof the fluid reservoirs 208 present on the tissue engineering module.Disassociated cells exit the digestion chamber 322 under the influenceof gravity sedimentation and/or fluid flow through theculture/proliferation chamber 300, and subsequently collect on theproliferation scaffold 310. Transfer of tissue aggregates out of thedigestion chamber 322 is precluded by the presence of a filtermembrane/mesh 316 a in the flow path between the digestion chamber 322and the culture/proliferation chamber 300. Following proliferation, thecells are released and transferred to the implantable differentiationscaffold 312 via the cell funnel 314. Again, membrane/mesh filters arepresent both upstream 316 b and downstream 316 c of the implantabledifferentiation scaffold 312 to ensure that the correct cell populationare seeded on the scaffold and that cells are not inadvertently lost towaste during fluid transfer operations.

In the preceding scenarios, various configurations of the proliferationsubstrate 310 or scaffold are possible, as illustrated for example inFIG. 12 b. For example, one configuration is a porous scaffold 310 ahaving a relatively even pore gradient. A pore gradient scaffold 310 bis a porous scaffold having a pore gradient where the pore sizedecreases as cells travel through the scaffold. This promotes a morehomogeneous distribution of cells throughout the scaffold at theconclusion of the cell seeding process. A pore gradient scaffold withreversed orientation 310 c may be used. Alternatively, a fiber filterscaffold 310 d, may be used which is a fibrous matrix typical of organiccompounds such as collagen. It is also possible to utilize a containedsuspension of micro-carriers (e.g. Cytodex™) as the proliferationsubstrate. Furthermore, the bioreactor may have an optical probe 324(shown in conjunction with the porous scaffold 310 a) supported by theCPU 128 to enable the inspection of the cell seeding process occurringwithin the proliferation scaffold and to further assess theproliferation events, particularly progress toward attaining a confluentcell layer.

As with proliferation, there are a variety of implantabledifferentiation scaffolds 312 that may be formed in differingconfiguration and of diverse materials (i.e. inorganic mineral-basedscaffolds such calcium phosphate, organic biopolymer scaffolds such ascollagen, etc.) and employed in the tissue engineering process. FIG. 12c illustrates a multi-zone differentiation/tissue formation chamber 306that comprises up to three implantable differentiation scaffolds 312,all of which may simultaneously proceed toward tissue constructformation. This allows for the preparation of different sizes ofimplantable tissue and for the use of alternate implantabledifferentiation scaffolds to assess and maximize tissue yield. Forexample, scaffold 312 a is a porous reticulate formed from a bonebiomaterial such as Skelite™ for use in bone and cartilage applicationswhere the tissue construct requires hard tissue anchoring within bone.The scaffold 312 a may be further enhanced through the use of a scaffoldmembrane/mesh 326 that encircles the implant to create a membraneencircled scaffold 312 b such that the loss of cells out of the scaffold312 a during the cell seeding process is minimized, thereby making thetissue engineering process more efficient. The membrane may preferablyonly partially encircle the scaffold or alternatively, more fullyencircle the scaffold. While the primary purpose of the scaffoldmembrane/mesh 326 is to contain the cells on and within the implantabledifferentiation scaffold 312, careful selection of the properties of thescaffold membrane/mesh 326 as is understood by one of skill in the arteither allows or limits the passage of specific molecular entities thatmay have a marked influence on the tissue engineering process at thecellular level.

A further embodiment is a gradient porosity and membrane encircledscaffold 312 c that combines the advantages of the scaffoldmembrane/mesh 326 with a pore gradient. The gradient is configured todeliberately cause the cells to collect on the top surface with onlyminimal propagation into the scaffold. A degree of porosity in thesurface is considered advantageous for tissue stability and for thesupply of nutrients to the developing tissue via the scaffold surface.This approach results in the development of a bipolar tissue constructwith distinct stratified zones. The top zone is essentially comprised ofde novo tissue. The bottom zone is essentially free of cells or tissueand remains as an open porous scaffold. The middle interfacial zonerepresents the structurally stable transition between the open scaffoldand the de novo tissue layer. Such a bipolar tissue construct is idealfor the repair of focal defects in articular cartilage as the top layeris tissue engineered cartilage that provides for lateral integrationwith the host cartilage, while the bottom layer provides for lateral andaxial integration with the subchondral bone. Integration of the bottomlayer with the surrounding subchondral bone may be further enhanced bythe application of bone marrow to the open scaffold at the time ofsurgical implantation. In cartilage repair applications, it is importantthat the mineral-based scaffold does not extend to the articularsurface, as this may compromise joint function. Accordingly, a secondarynon-mineral scaffold (not shown in the figures) may be employed in thetop zone of de novo cartilage to assist with the formation of tissueconstructs of sufficient size to treat large cartilage lesions (i.e. upto 10 cm² in diameter and 2-3 mm in thickness). Furthermore, thesecondary scaffold can be configured to generate shaped constructs thathave articular surface profiles that more closely match the particularanatomical characteristics present at the site of implantation.Candidate materials for the secondary scaffold are synthetic biopolymers(e.g. PGA, PLA) or natural biopolymers (e.g. alginate, agarose, fibrin,collagen, hyaluronic acid). These secondary scaffolds may be in the formof hydrogels or three-dimensional preformed scaffolds.

Alternate techniques for the preparation of bipolar tissue constructsare possible within the tissue engineering system. The implantabledifferentiation scaffold 312 may be partially infiltrated with abioresorbable polymer that limits cell seeding to certain regions of thescaffold. This creates a preferential zone of new tissue formationduring the preparation of the tissue construct. Upon implantation, thepolymer is resorbed thereby leaving voids in the porous scaffold thatpromote anchorage within the host tissue. A further configurationinvolves an implantable scaffold with relatively open porosity that ispositioned away from the exit of the differentiation/tissue formationchamber. During cell seeding, this open space provides for thecollection of cells that migrate through the open scaffold. As cells areaccumulated within the differentiation/tissue formation chamber, boththe open space and a portion of the scaffold become infiltrated withcells and thereby create a preferential zone of new tissue formation.The resulting tissue construct comprises a de novo tissue zone that isdevoid of the scaffold, a middle transition zone or interfacial zonecontaining both de novo tissue and the scaffold, and a region of theporous scaffold that is open and essentially free of cells or tissue.

FIG. 12 d illustrates a bioreactor monitoring scheme whereby sensors 216(i.e. temperature, pH, dissolved gases, etc) are integrated into the lid204 of the bioreactor 202 to provide feedback to the CPU 128 of theprogress of the tissue engineering process. In addition, a CCD camera330 may be employed to monitor the optical properties of theproliferation scaffold 310 (or substrate) for evidence of impendingconfluence (e.g. optical density and/or light scattering as a functionof cell density) such that cell release is timed to maximize the cellyield from the proliferation step.

FIG. 13 better shows the flow path and fluid circulation within thebioreactor 202. The bioreactor 202 is shown to have an inlet port 302,an outlet port 304 and an internal cavity defining a basic chamber 300.Fluid flows from the fluid flow plate 220 into the bioreactor 202 viathe inlet port 302 and exits through the outlet port 304. The bioreactorlid 204 attaches to the bioreactor 202. A variety of different mountinghardware may be used to hold the bioreactor lid 204 and bioreactor 202together. The chamber 300 may be designed to accommodate one or moresubstrates or scaffolds 310. Furthermore, the bioreactor 202 may besubdivided into separate chambers that permit the steps of tissuedigestion, proliferation, differentiation, and tissue formation. Eachchamber may be configured with inlet and outlet ports that areindependently controlled via flow control valves for greater controlover the tissue engineering sequence. Circulation of fluid is effectedby the activation of one or more flow control valves 214 and the pumpunit 122, based on control signals from the CPU 128. Depending upon thespecific valves activated, operation of the pump unit 122 moves fluidfrom one of the fluid reservoirs 208 into the bioreactor 202 or permitsrecirculation of the fluid within the bioreactor. For biologicalprocesses that require stable dissolved gas concentrations,recirculation is advantageous as it enables the fluid to be passedacross a membrane that facilitates gas exchange. The nature of theexchange is based on the dissolved concentrations in the bioreactorversus the external conditions established by the ECU 136. Thebioreactor lid 204 is shown to have a sampling port 332 and sensorprobes 216 that are operably connected to the interior chamber of thebioreactor. Alternatively, the sampling port may be provided elsewhereon the bioreactor housing. The sampling port allows the removal oraddition of materials into and out from the bioreactor. The samplingport may be replaced or augmented with a gas exchange membrane asrequired.

FIG. 14 illustrates a multi chamber bioreactor 202 with the bioreactorlid 204 removed for clarity. The inlet port 302 is connected to a tissuedigestion chamber 322. The configuration of the tissue digestion chamber322 permits a patient biopsy to be loaded into the bioreactor forsubsequent automated digestion to yield disassociated cells. Thecirculation path within the digestion chamber promotes the gentleagitation of the biopsy to prevent stagnant areas that could potentiallylead to excessive exposure of the biopsy tissue to circulating digestionenzymes. Furthermore, the inlet and/or outlet of the digestion chambermay house a filter membrane/mesh 316 (not shown) of varying porosity toprovide for cell sorting and to preclude the release of partiallydigested tissue aggregates. The bioreactor contains a second chamber 300that accommodates a proliferation substrate or scaffold 310 forreceiving cells for proliferation. The proliferation scaffold may beformed in various geometries that support both two-dimensional andthree-dimensional proliferation, and may be comprised of variousbiocompatible materials that promote cell proliferation, such as calciumphosphate biomaterials (for example Skelite™), biopolymers, or naturalmatrices (for example collagen). Cells delivered from the tissuedigestion chamber 322, or via the optional cell inoculation port 334,become dispersed on or within the proliferation substrate or scaffold310 and proliferate, thereby increasing the cell population forsubsequent cell differentiation and tissue formation. Note that theprocess may be terminated following proliferation if the goal is to onlyexpand the cell population without further differentiation. Animplantable differentiation scaffold 312 is present within adifferentiation/tissue formation chamber 306 at the base of thebioreactor 202. As with the proliferation scaffold 310, the implantabledifferentiation scaffold 312 may be formed in different geometries andmay be composed of a variety of biocompatible materials that areproperly selected to meet the biological requirements of the implantsite, (for example Skelite™ is an ideal candidate implant for skeletalsites).

In operation, cells are released from the proliferation substrate orscaffold 310 through an automated sequence, such as the delivery ofenzymes (for example trypsin) and the timed application of impact to thebioreactor via the impact drive 266 (not shown). The cell suspensionmigrates under the controlled flow conditions present in the bioreactorinto the implantable scaffold 312 via the cell funnel 314, whereupon thecells become resident and initiate the differentiation and tissueformation sequence. Upon conclusion of this sequence, the tissue soformed may be removed from the bioreactor for subsequent implantation.One skilled in the art would understand that the particular embodimentof the bioreactor of FIG. 14 is only a representative design example.The bioreactor, in general, can be configured in various ways withrespect to overall shape, size and internal configuration withoutadverse effect on function. For example, a gas exchange membrane 336present on the bioreactor may be a separate and discrete component thatis connected in-line with one or more fluid delivery tubes 210 or theflow plate 220 of the tissue engineering module 118. Furthermore, thechambers of the bioreactor may be isolated from each other via controlvalves to avoid the necessity for all fluids to pass through allchambers. When required the passageways between chambers may be openedto effect the transfer of fluids and cell suspensions. An example ofsuch a variation that enables increased flexibility in bioprocessingconditions and sequences is illustrated in FIG. 17. An alternateconfiguration to enable controlled exposure of the implantabledifferentiation scaffold 312 to the contents of the bioreactor is theuse of a shuttle 318 that isolates the implantable scaffold until cellseeding is required as part of the differentiation step. To enable cellseeding, the shuttle 318 moves the implantable scaffold into the fluidflow from a protected location within the bioreactor. Variousconfigurations of the shuttle are possible, including rotation-basedmovement or the use of a removable barrier that isolates the implantablescaffold until cell seeding is required.

FIG. 15 illustrates a rotational bioreactor design that takes advantageof the orientation of the gravity vector to effect cell transport bysedimentation at different stages in the tissue engineering process.Note that while this figure illustrates rotation of the bioreactor, thetechnical objective may be equally attained by rotation of the tissueengineering module or indeed by rotation of the entire housing 102. Asshown in FIG. 15, the bioreactor 202 is attached to a rotational shaft350 which is affixed to the structural spine 200 of the tissueengineering module 118. This provides a mechanism for the rotation ofthe bioreactor 202 in order that cell seeding via sedimentation canoccur on to selected proliferation surfaces within theculture/proliferation chamber 300. The proliferation surfaces of thebioreactor may be optionally coated with biomaterials that enhanceproliferation (for example Skelite™), or a dedicated proliferationsubstrate or scaffold may be inserted into the chamber 300 to providethis role. As an alternative to the use of the digestion chamber 322, asecond inoculation port 352 is provided at the side of the bioreactor202 to enable direct cell seeding. Cells may be initially seeded on aproliferation surface 354 which is relatively small (FIG. 15 a). Basedon elapsed proliferation time or the detection of confluence, the cellsmay be automatically released and the bioreactor rotated via therotational shaft 350 such that the cells released from the proliferationsurface 354 will sediment on to the increased area of surface 356 (FIG.15 b), allowing further proliferation. At the completion of thesecondary proliferation step, the expanded cells are released and thebioreactor is again rotated to permit seeding of the implantablescaffold 312 (FIG. 15 c). Thus the rotational shaft 350 and associatedflexible tubes 358 allow the bioreactor 202 to be rotated as required tomaximize the use of gravity sedimentation in sequential proliferationstages. It is within the scope of the present invention to use therotational shaft in a manner to agitate or shake the bioreactor wheresuch conditions are desirable.

Referring now to FIG. 16, the tissue engineering module 118 may beadapted to include techniques for the sterile sampling of suspendedcells, tissue culture fluids, and/or waste products. In this embodiment,a syringe manifold 400 and sterile offloading ports 402 are integratedinto the structural spine 200 of the tissue engineering module 118.Microbore tubing 406 links the syringe manifold to the bioreactor 202via the sampling port 332. Syringes 404 are connected to offloadingports 402 at the manifold 400 to enable the collection and removal offluid samples or cell suspensions for subsequent analysis withoutcompromising the operation, integrity or sterility of the tissueengineering process. An alternate sampling technique is also providedwhereby a fusible bioreactor sampling line 408 is connected to thebioreactor lid 204. As this line is physically linked to the interior ofthe bioreactor and is in close proximity to the biological eventsunderway therein, the line contains fluid of substantially the samecomposition as that present within the bioreactor. Consequently, arepresentative sample of the bioreactor fluid may be obtained by fusingthe ends of the sampling line and then removing the line from the tissueengineering module for subsequent analysis. It will be obvious to oneskilled in the art that such a fusible line can be used as the basis fora sampling technique through the automatic operation of sealingcomponents within the housing 102.

FIG. 17 illustrates a more complex fluid flow schematic for the tissueengineering module 118 in which the different requirements fordigestion, proliferation and differentiation are accommodated byseparate bioreactor chambers. These chambers may be present within aseries of discrete bioreactors or combined within a single bioreactorthat maintains separate control over the conditions in each chamber. Atissue digestion chamber 322 is present that accommodates a tissuebiopsy 320. A proliferation chamber 300 is present that is configured toaccept cells from the digestion chamber 322 and allows seeding of aproliferation substrate or scaffold 310. A differentiation/tissueformation chamber 306 is also present that is configured to accept theexpanded cell numbers from the proliferation chamber 300 and allowsseeding of an implantable scaffold 312.

Tissue engineering reagents (i.e. media, enzyme solutions, washingsolutions, etc.) are loaded in fluid reservoirs 208 a-208 e. Wasteproducts are collected in fluid reservoir 208 f, which can be manuallyaspirated for sampling purposes using access port 212f. Additional fluidreservoirs may form part of the fluid reservoir system 206 and beaccommodated on the tissue engineering module as required for differenttissue engineering processes. Fluid flow through the system is directedby the operation of fluid pumps 122 a-122 k, flow control valves 214a-214 c, and uni directional flow valves 410 a-410 c (i.e. fluid flowcheck valves). Furthermore, pumps 212 a-212 k are configured to operateas active pumps or passive valves (open/closed), according to controlinputs from a central microprocessor. Filters 316 a-316 d are used toselectively control the movement of cell suspensions within the systemand to limit the passage of cell aggregates during washing andtransition stages of the tissue engineering process. Levels of dissolvedgasses in the media are maintained via the in-line gas exchangemembranes 282 a and 282 b. Optional syringes 404 a and 404 b are presentto allow cell collection or media sampling via sterile offloading ports402 a and 402 b.

In operation, a tissue biopsy 320 is inserted into the tissue digestionchamber 322 between filters 316 a and 316 b. A digestion mediumcontaining enzymes is pumped into the tissue digestion chamber 322 fromthe fluid reservoir system 206 to initiate the digestion process.Digestion may be enhanced by gentle agitation of the digestion mediumwithin the digestion chamber via a mixing diaphragm to maximize reagentexposure to the biopsy. The digestion medium may be continuously orperiodically re-circulated via pump 122 g. During recirculation, thefluid flow is directed into the bottom of the digest chamber, againstthe gravity vector, in order to suspend and tumble the tissue biopsy,thereby maximizing the effectiveness of the digestion process. Filter316 a prevents migration of cells and cell aggregates into the fluidpathway. The recirculation path includes the in-line gas exchangemembrane 282 a which provides for consistent levels of dissolved gasesin the digestion medium. Introduction of a washing solution, containedin the fluid reservoir system 206, into the bottom of the digestionchamber 322 flushes the digestion chamber and effectively washes thedigestion medium from both the disassociated cells and any residual cellaggregates. Following a single or multiple washing procedures, theapplication of reverse flow transfers the cell suspension to either theproliferation chamber 300 or the optional syringe 404 a for externalinspection or analysis. The transfer of partially digested tissue out ofthe digestion chamber is precluded by filter 316 b that is sized toallow passage of disassociated cells and retention of cell aggregates.

Cells generated from the biopsy digestion process or available viadirect loading of a cell suspension are seeded through fluid flow and/orgravity sedimentation onto a proliferation substrate or scaffold 310present within the proliferation chamber 300. Following a quiescentperiod to allow attachment of the cells to the proliferation substrateor scaffold 310 (for the example of attachment dependent cells), aproliferation medium is introduced into the proliferation chamber 300from the fluid reservoir system 206. This medium is periodicallyreplaced with fresh proliferation medium from the reservoir system 206at specific times during the proliferation phase. In between the mediumreplacement steps, the fluid within the proliferation chamber iscontinuously or periodically recirculated under the control of pumps 122g, 122 h and 122 i, plus control valves 214 a and 214 b. The fluiddelivery and recirculation paths include the inline gas exchangemembrane 282 a which provides for consistent levels of dissolved gasesin the proliferation medium. During a medium replacement step, thesupply of fresh medium from the fluid reservoir system 206 is balancedby the removal of fluid to the waste reservoir 208 f via pump 122 f.Thus, through a combination of periodic medium replacement steps andcontrolled recirculation, the tissue engineering system maintainsoptimal conditions within the proliferation chamber throughout theproliferation process.

Once the cell culture approaches confluence, the media within theproliferation chamber 300 is evacuated into the waste reservoir 208 f bypump 122 f. In this process, the removal of fluid from the proliferationchamber is balanced by incoming sterile air delivered via a sterilefilter port on the proliferation chamber (not shown) or by incoming PBSwash solution from the fluid reservoir system 206. The cells are washedextensively by two consecutive washing steps with the PBS wash solutionto remove residual proliferation medium. The cells are subsequentlyreleased from the proliferation substrate or scaffold 310 through anautomated sequence, such as the delivery of enzymes (for exampletrypsin) and the timed application of impact to the bioreactor via animpact drive. Following cell release, the enzymatic process may bestopped by the delivery of media containing serum that inhibits enzymeactivity. In order to collect the cells for eventual seeding on to theimplantable scaffold 312 within the differentiation/tissue formationchamber 306, the cell suspension is transferred from the proliferationchamber 300 to the filter 316 c. The filter 316 c prevents the passageof cells but allows the media to continue via valve 214 b to the wastereservoir 208 f under the control of pump 122 f. The collected cells arethen released from the filter 316 c by the application of reverse flowand are delivered to either the differentiation/tissue formation chamber306 or the optional syringe 404 b for external inspection or analysis.

Cell seeding on to the implantable differentiation scaffold 312 isachieved by transferring the cells from the filter 316 c to the topsurface of the scaffold via pump 122 j. The loss of cells away from thescaffold is minimized by the optional use of a scaffold membrane or mesh326. Following cell seeding, fresh differentiation media may beintroduced into the differentiation/tissue formation chamber 306 througha secondary input by the operation of pump 122 k. This secondary inputis located away from that region of the implantable scaffold that isseeded with cells so as to minimize the potential for damaging sheerstresses that could compromise the formation of cell aggregates. Thedifferentiation medium is periodically replaced with freshdifferentiation medium from the reservoir system 206 at specific timesduring the differentiation phase. In between the medium replacementsteps, the fluid within the differentiation/tissue formation chamber iscontinuously or periodically recirculated under the control of pumps 122j or 122 k, plus control valve 214 b. The path for the delivery of bothfresh differentiation medium and recirculated medium includes thein-line gas exchange membrane 282 b which provides for consistent levelsof dissolved gases in the differentiation medium. During a mediumreplacement step, the supply of fresh medium from the fluid reservoirsystem 206 is balanced by the removal of fluid to the waste reservoir208 f via pump 122 f. Environmental conditions within thedifferentiation/tissue formation chamber are monitored and controlledfor the period necessary for the successful formation of the tissueconstruct, at which time the differentiation/tissue formation chamber ofthe bioreactor is opened and the construct retrieved for subsequentclinical or research use.

FIG. 18 illustrates a variation on the fluid flow schematic of FIG. 17where the proliferation scaffold or substrate 310 within theproliferation chamber 300 is replaced with a planar proliferationsubstrate of relatively large surface area. The orientation of thesubstrate is such that cell sedimentation under gravity evenlydistributes the cells over the proliferation surface. Provided that thecorrect orientation of the proliferation chamber is maintained, theproliferation substrate may be in the form of a rigid polymer cultureplate or a flexible wall container.

FIG. 19 shows a tissue digestion bioreactor 500 that contains a tissuedigestion chamber 322 of an appropriate size to accommodate one or moretissue samples such as a tissue biopsy 320. The bioreactor 500 consistsof four primary components: a bioreactor base 502 that substantiallyforms the tissue digestion chamber 322, a removable bioreactor lid 504,port filter 316 b, and optional port filter 316 a (not shown).

The bioreactor lid 504 provides for a media port 506 with an optionalport filter 316 a (not shown) and an air outlet port 508. The bioreactorbase 500 accommodates filter 316 b that allows passage of disassociatedcells out of the tissue digestion chamber 322, via media port 510, andretention of tissue aggregates and biopsy debris.

Following insertion of the tissue biopsy 320, the bioreactor is filledunder automated control with an enzyme solution through port 506 or port510. The addition of enzyme solution to the tissue digestion chamber 322is balanced by air escaping through port 508. Biopsy digestion takesplace under continuous or intermittent recirculation of the enzymesolution, thereby keeping the released cells in suspension andmaximizing the exposure of the biopsy to the enzyme reagents. Duringrecirculation, the enzyme solution enters the bioreactor through port510 and leaves via port 506. This creates a fluid flow path in adirection opposite to the gravity vector such that the biopsy issuspended and tumbled to maximize the effectiveness of the enzymereagents. Digestion may be enhanced by gentle agitation of the digestionmedium within the digestion chamber via a mixing diaphragm (not shown).Port 508 may be closed during any recirculation steps, as air bubblespresent in the fluid flow system are trapped in the upper half of thebioreactor, above the inlet 512 of port 506. Upon completion of thedigestion sequence, the application of reverse flow of either air ormedium through port 506 transfers the disassociated cells through port510 to either a proliferation chamber or a cell collection vessel.

FIG. 20 shows a proliferation bioreactor 520 that provides for aproliferation chamber 300. The bottom of the proliferation chamberconsists of proliferation substrate 310 suitable for cell attachment andgrowth. To adjust or maintain the levels of dissolved gases in themedium, a gas permeable membrane (not shown) may be incorporated to thetop surface of the proliferation chamber that allows the transport ofgases such as oxygen and CO₂. Separation walls 522 divide the internalspace of the proliferation chamber into a channel system that forcesmedium to follow a predefined pathway from the inlet port 524 to theoutlet port 526.

The design of the proliferation bioreactor design has several importantoperational features. Relatively uniform cell seeding can be obtained bythe infusion of a cell suspension through the channel system.Furthermore, the channel configuration ensures that media flow is welldistributed over the whole proliferation surface, thereby reducingpotential low-flow regions that may compromise local cell vitality dueto reduced nutritional supply or waste product removal. At theconclusion of the proliferation sequence, continuous or intermittentrecirculation of an appropriate enzyme solution through the channelsystem induces uniform cell detachment due to the effect of the enzymereaction and the low-level sheer stresses generated by the fluid flow.Accordingly, cell harvest is achieved without the need for mechanicalshaking or rotation of the proliferation chamber.

FIG. 21 shows a differentiation bioreactor 530 designed to promote celldifferentiation and subsequent tissue construct formation. Thebioreactor consists of four primary components: a bioreactor base 532that substantially forms a differentiation/tissue formation chamber 306,a removable bioreactor lid 534, a permeable membrane tube 326, and adifferentiation scaffold 312. The permeable membrane tube 326 tightlyencircles the scaffold reticulate to form a tissue growth compartment536 above the scaffold. The tissue growth compartment may extend withinthe scaffold according to the pore size of the scaffold and theplacement of the scaffold within the membrane tube. The membrane tube isalso affixed to the inlet 540 of port 542, such that the membrane isphysically located centrally within the differentiation/tissue formationchamber 306. This divides the bioreactor into two independentcompartments, a cell and tissue growth compartment 536 and an outercell-free medium compartment 538, all within the overalldifferentiation/tissue formation chamber 306. The pore size of themembrane tube is selected on the basis of being impermeable for cellsbut permeable for nutrients, waste products, growth factors, etc.,within the culture medium. If desired, membrane pore size can be chosenin a manner to exclude molecules of a certain molecular weight frompassing through the membrane.

The bioreactor lid 534 has two air outlets ports 542 and 544, and onemedia inlet port 546. The bioreactor base 532 accommodates two furtherports 548 and 550. The inlet port 546 is required for loading a cellsuspension into the tissue growth compartment 536 and for the perfusionof the emerging tissue construct with culture medium. During thedelivery of the cell suspension into the empty tissue growthcompartment, entrapped air is allowed to exit through port 542. In asimilar fashion, the outer cell free compartment 538 is loaded withmedia via port 548 or port 550 and entrapped air may escape via port544.

The design of the differentiation bioreactor allows direct perfusion ofthe tissue construct through media delivery to port 546 or indirectmedia supply to the surrounding cell free compartment 538 via port 548.Typically, ports 542 and 544 are closed during perfusion and port 550serves as a media outlet; however, various alternate media supplyscenarios are possible based on specific tissue engineeringrequirements. An important aspect of the media perfusion strategy isthat the permeable membrane 326, which forms part of the tissue growthcompartment, allows fresh culture medium to permeate into the tissuegrowth compartment without any loss of cells away from the scaffold.Furthermore, nutrition is provided to the cells from essentially alldirections without restrictions from any impermeable bioreactor walls.

FIG. 22 illustrates a further embodiment of the fluid flow schematic inwhich the bioreactors of FIGS. 19-21 may be employed. A tissue digestionchamber 322 is present that accommodates a tissue biopsy. Aproliferation chamber 300 is present that is configured to accept cellsfrom the digestion chamber 322 and allows seeding of a proliferationsubstrate. A bubble trap 560 removes air bubbles from the input line tothe proliferation chamber and therefore prevents these bubbles fromentering the proliferation chamber 300 and potentially compromisinglocalized cell populations. A reservoir 562 is present to accept theexpanded cell numbers from the proliferation chamber 300 and to serve asa temporary holding container during a cell washing and cellconcentration procedure performed with the aid of a cross flowfiltration module 564. A differentiation/tissue formation chamber 306 isalso present that is configured to accept the cells from reservoir 562after the washing and concentration step and allows seeding of animplantable scaffold 312.

Tissue engineering reagents (i.e. media, enzyme solutions, washingsolutions, etc.) are stored in fluid reservoirs 208 a-208 e. Wasteproducts are collected in fluid reservoir 208 f. Fluid flow through thesystem is directed by the operation of fluid pumps 122 a and 122 b, flowcontrol valves 214 a-214 v according to control inputs from a centralmicroprocessor. Air filters 566 a-566 c allow the transfer of air intoor out of the system as required during operation without compromisingsystem sterility. Furthermore, in-line gas exchange membranes (notshown) may be deployed at various locations within the fluid flow pathsto facilitate the control of dissolved gases in the culture medium.

In operation, a tissue biopsy 320 is inserted into the tissue digestionchamber 322. A digestion medium containing enzymes is pumped into thetissue digestion chamber 322 from a fluid reservoir 208 to initiate thedigestion process. The digestion medium may be continuously orperiodically re-circulated via pump 122 a, thereby keeping the releasedcells in suspension and maximizing reagent exposure to the biopsy.Introduction of a proliferation culture medium from one of the fluidreservoirs 208 into the top of the digestion chamber 322 transfers thecell suspension to the proliferation chamber 300 and simultaneouslydilutes the enzyme solution to a concentration that is tolerable forcell proliferation in the in the proliferation chamber 300. The transferof partially digested tissue out of the digestion chamber is precludedby port filter 316 b that is sized to allow passage of disassociatedcells and retention of cell aggregates. Cells generated from the biopsydigestion process are homogeneously distributed throughout theproliferation chamber 300 either by the recirculation of the cellsuspension via the activation of valves 214 h, 214J, 214 l and the pump122 a, or by the automated application of gentle shaking of theproliferation bioreactor.

Following a quiescent period to allow attachment of the cells to theproliferation substrate, the proliferation medium is periodically orcontinuously replaced with fresh proliferation medium from one of thefluid reservoirs 208. During a medium replacement step, the supply offresh medium from the fluid reservoir system 208 is balanced by theremoval of fluid to the waste reservoir 208 f via valve 214 i.

Once the cell culture approaches confluence, the media within theproliferation chamber 300 is evacuated into the waste reservoir 208f. Inthis process, the removal of fluid from the proliferation chamber isbalanced by incoming sterile air delivered via a sterile filter 566 a orby incoming PBS wash solution from one of the fluid reservoirs 208.

The cells are subsequently released from the proliferation substratethrough an automated sequence, such as the delivery of enzymes (forexample trypsin) and the timed recirculation of the cell suspension orthe timed application of impact or agitation to the bioreactor via animpact drive. In order to remove the enzymes and to collect the cells ina relatively small volume of medium for subsequent transfer to the celldifferentiation chamber 306, the cell suspension is transferred from theproliferation chamber 300 to the reservoir 562. The cell suspension isthen continuously recirculated via valves 214 m, 214 j, 214 q and pump122 a through the cross-flow filtration module 564. The membrane in thecross flow filtration module 564 prevents the loss of cells but allows acertain percentage of media (permeate) to be removed via valve 214 o tothe waste reservoir 208 f. The consequence is a reduction of thesuspension volume and/or dilution of any enzymes present, provided theremoval of permeate is compensated by the supply of fresh medium fromone of the fluid reservoirs 208. The continuous flow reduces thepotential for cells to become entrapped within the membrane of thecross-flow module 564.

Cell seeding on to the implantable differentiation scaffold 312 isachieved by transferring the washed cells from the reservoir 562 to thetop surface of the scaffold via the valves 214 m, 214 j, 214 p, and pump122 a. The loss of cells away from the scaffold is minimized by theoptional use of a scaffold membrane or mesh 326. Following cell seeding,fresh differentiation media may be introduced into thedifferentiation/tissue formation chamber 306 through the operation ofpump 122 b. The differentiation medium is periodically or continuouslyreplaced with fresh differentiation medium from the reservoir system.During a medium replacement step, the supply of fresh medium from one ofthe fluid reservoirs 208 is balanced by the removal of fluid to thewaste reservoir 208 f via valve 214 u. In between the medium replacementsteps, the fluid within the differentiation/tissue formation chamber iscontinuously or periodically recirculated under the control of pump 122b, valve 214 t, and either valve 214 r for perfusion through the tissueconstruct or valve 214 s for delivery outside the scaffold membrane 326.This secondary fluid delivery path outside the scaffold membrane islocated away from that region of the implantable scaffold that is seededwith cells so as to minimize the potential for damaging sheer stressesthat could compromise the formation of cell aggregates. As with theprevious embodiments of the fluid flow schematic, environmentalconditions within the differentiation/tissue formation chamber aremonitored and controlled for the period necessary for the successfulformation of the tissue construct, at which time thedifferentiation/tissue formation chamber of the bioreactor is opened andthe construct retrieved for subsequent clinical or research use.

FIG. 23 illustrates an embodiment of the invention where the tissueengineering module as described herein comprises three bioreactors. FIG.23 illustrates the combined use of the tissue digestion bioreactor ofFIG. 19 having an internal tissue digestion chamber 322, with theproliferation bioreactor of FIG. 20 having a proliferation chamber 300,and the differentiation bioreactor of FIG. 21 having a differentiationchamber 306. These bioreactors are operably connected on a tissueengineering module to provide for the automated steps involved in thesequence of tissue digestion, cell proliferation, cell differentiation,and tissue formation.

It is understood by one of skill in the art that the automated tissueengineering system may comprise one or more bioreactors as supported toa housing either by a structural support or by equivalent means. Whencomprising two or more bioreactors, the bioreactors may be operativelyconnected or alternatively, independently operable and/or co-operativelyoperable. Furthermore, each bioreactor may comprise a different internalchambers or the same type of chambers. In a further embodiment, thechambers and/or bioreactors are operably connected to provide for theexchange of fluids, cells and/or tissues between the chambers and/or thebioreactors.

The automated tissue engineering system of the invention is easy toprepare for use. The following sequence is a representative example forthe preparation of a cartilage implant based on the use of the tissueengineering system of the present invention for the repair of focaldefects in articular cartilage. For this application, the stages oftissue digestion, cell proliferation and cell differentiation/tissueformation are required. The three stages of the tissue engineeringprocess may be accomplished by way of a single bioreactor with multiplechambers or three separate and discrete bioreactors, as shown in FIGS.17, 18 and 22.

Prior to initiating the tissue engineering sequence, the followingreagent compositions are loaded into the reservoirs 208 a through 208ein the tissue engineering module via the reservoir injection ports 212.Reagent A is utilized for the digestion of chondrocytes derived fromsmall human articular cartilage biopsies. Reagents B, D and E areutilized for cell proliferation. Reagent C is utilized fordifferentiation and tissue construct formation.

-   -   Reagent A—Digestion Medium: DMEM/F-12, 5% FCS or autologous        serum, 1 μg/ml Insulin, 50 μg/ml Ascorbic Acid, 100 IU/100 μg/ml        Pen/Strep, 2.5% Hepes Buffer, 0.1% (1 mg/ml) Pronase and 0.025%        (0.25 mg/ml) Collagenase, pH 7.4    -   Reagent B—Proliferation Medium: DMEM/F-12, 10% FCS or autologous        serum, 10 μg/ml Ascorbic Acid, 100 IU/100 μg/ml Pen/Strep, 2.5%        Hepes Buffer, pH 7.4    -   Reagent C—Differentiation Medium: DMEM/F-12, 10% FCS or        autologous serum, 1 μg/ml Insulin, 50 μg/ml ascorbic acid, 100        IU/100 μg/ml Pen/Strep, 2.5% Hepes Buffer, pH 7.4    -   Reagent D—PBS Wash Solution: 137 mM NaCl, 3.7 mM KCl, 8 mM        Na₂HPO₄*2H₂O, 1.5 mM KH₂PO₄, in H2O, pH 7.4    -   Reagent E—Cell Release Solution: 1× Trypsin solution

The above reagents are nominally stable for periods up to several weekswhen stored at 4° C. on the tissue engineering module within the systemenclosure. Enzymes may be stored lyophilized within the tissueengineering module and hydrated at the time of use. This allows customenzyme tailoring to the specific tissue engineering application.

A human cartilage biopsy (100-500 mg) is obtained through anarthroscopic surgery from a non-load bearing area on the upper medialfemoral condyle. Prior to loading the biopsy into the digestion chamber,the biopsy is weighed and the mass recorded for subsequent data entryinto the programming sequence for the base unit. Following massdetermination, the biopsy is placed within the digestion chamber and thebioreactor is closed ready for the tissue engineering module to beinserted into the base unit of the tissue engineering system. Once thetissue engineering module is installed, the CPU of the base unit is thenprogrammed via the user interface according to the size of the biopsyand the tissue engineering sequence desired.

On initiation of the programmed automated sequence, pronase/collagenasedigestion of the biopsy is commenced by an infusion of Reagent A intothe digestion chamber of the bioreactor through the activation of therequired flow valves and the operation of the fluid delivery pump.Digestion is performed at 37° C. over a 16 hour period under continuousor intermittent recirculation of Reagent A to keep cells in suspensionand to to maximize reagent exposure to the biopsy. This may be followedby two consecutive washing steps in Reagent D. At the end of thisdigestion sequence, approximately 200,000 to 500,000 cells per 100 mg ofbiopsy tissue are obtained.

At this point a sample of the digested cells may be retrieved via thesampling port in order to assess cell number and vitality. Thisbiological assessment is typically assessed outside the system by way ofa hemocytometer after staining with trypan blue.

Under the automated control of the base unit, the disassociated cellsare delivered on to the proliferation substrate or scaffold present inthe proliferation chamber of the bioreactor in order to establish a cellseeding density between 2000 cells/cm² and 15000 cells/cm². To effectcontinued proliferation toward confluence, Reagent B is supplied from areservoir on the tissue engineering module according to a preprogrammedflow profile. The temperature and pH of the medium are monitored todetect deviations from 37° C. and pH 7.4, respectively. In addition, thestatus of cell proliferation is indirectly assessed by detection ofmetabolic turnover as a function of time (e.g. pH, O₂, CO₂, lactic acidand glucose consumption). The level of confluence is further supportedby optical monitoring via CCD camera linked to the proliferation probeembedded within the proliferation chamber. Once impending confluence isdetermined either empirically or by way of sensor-based monitoring, thecells are washed extensively by two consecutive washing steps withReagent D to remove all culture medium.

Detachment of propagated cells from the proliferation substrate orscaffold is initiated by the transfer of Reagent E from a reservoirwithin the tissue engineering module into the proliferation chamber.This trypsin solution is present for 5 minutes within the bioreactorwhereupon the reaction is stopped by the automatic addition of Reagent Bwhich contains FCS or autologous serum that inhibits enzyme activity.Cell release from the proliferation substrate or scaffold is furtherenhanced by the application of low frequency impact to the bioreactorvia the impact drive or recirculation of the trypsin solution. Oncereleased, a cell washing and filtration step is performed in order toremove the trypsin and to concentrate the cell suspension for subsequenttransfer on to the scaffold present in the differentiation/tissueformation bioreactor.

For this application, a bipolar configuration is ideal as this providesfor cartilage layer at the articular surface that is connected to aporous scaffold layer, formed of a bone biomaterial such as Skelite™,for integration with the subchondral bone. The preparation of thebipolar construct may be achieved through one of several alternateprocedures. The differentiation scaffold may be formed with a poredensity gradient that preferentially traps cells at one end creating aregion of high cell concentration which promotes the formation of thecartilage layer. Alternately, the scaffold may be previously coated onone end with fibrin gel to preclude cell attachment and cartilage matrixformation in this region. With either approach, the loss of cells awayfrom the scaffold is minimized by the optional use of an encirclingmembrane or mesh. The flow rate for cell delivery is low to ensure fluidshear does not damage the proliferated cell population. Following thecompletion of the cell seeding step, fluid flow through thedifferentiation/tissue formation chamber is stopped to enable theformation of cell aggregates, as this is known to be crucial in terms ofsuccessful differentiation. Following this important step, perfusion ofReagent C is performed over the period necessary for tissue formationand maturation in order to optimally supply cells with nutrients and toremove waste products. After this culture period, the cells will haveproduced extracellular matrix that is substantially identical to that ofnative human articular cartilage. The properties of the tissue formedcan be confirmed by independent external biochemical methods such ascollagen typing via SDS-PAGE and gene expression. As a final step in theprocess, the tissue engineering system provides notification by way ofthe user interface that the sequence is complete and the tissueengineering module may be removed to harvest the implant. The tissueengineering module or a detachable form of the bioreactor may betransported to the operating room whereupon the bioreactor lid isremoved in a sterile field and the implant retrieved for surgical use.

It should be noted that the system of the invention is not limited to aparticular type of cell or tissue. For example, a skeletal implant maybe prepared for use in the reconstruction of bone defects. In thisapplication, bone marrow could be used as the source of the primaryand/or precursor cells required for the tissue engineering process.Accordingly, there is no requirement to perform tissue digestion; hence,the bioreactor may be of the type that only supports proliferation anddifferentiation. Depending on the available cell population and therequired size of the implant, even proliferation may not be required. Inthis case, the configuration of the bioreactor may be directed to thesingle stage of cell differentiation and ongoing tissue formation. Thefinal tissue construct would be comprised of an implantable scaffold,which may be composed of a bone biomaterial such as Skelite™, withactive bone cells lining the open pores of the scaffold and activelylaying down new mineralized matrix (osteoid). Such an implant would bequickly integrated at the implant site thereby accelerating the recoveryprocess.

As a further example of the flexibility of the system, tissue engineeredblood vessels may be prepared using culture expanded endothelial cellsseeded onto flexible scaffolds of a tubular geometry in the finaldifferentiation stage.

The integrated tissue engineering system of the present invention hasseveral advantages compared to methods and systems of the prior art. Inparticular, the turnkey operation of the device enables complex tissueengineering procedures to be performed under automated control in theclinic, thereby precluding the need to transport cells to centralizedfacilities for biological processing. The system is simple to use andobviates the existing time consuming and expensive human tissue cultureprocedures which often lead to implant contamination and failure. Thetissue engineering modules and associated subsystem assemblies may becustomized for the type of cell or tissue to be cultured and may befabricated from any suitable biocompatible and sterilization tolerantmaterial. The entire tissue engineering module or specific componentsthereof are replaceable and may be considered disposable. The tissueengineering module may be provided in a single-use sterile package thatsimplifies system set-up and operation in clinical settings.

It is understood by those skilled in the art that the tissue engineeringmodule and device of the present invention can be fabricated in varioussizes, shapes and orientation. The device can be fabricated toincorporate a single tissue engineering module or multiple modules invertical or horizontal formats. Accordingly, the subassemblies can bemade to correspond to the spatial format selected for the tissueengineering device. As such, different types of tissue engineering canbe simultaneously conducted in a single device with each tissueengineering sequence being automatically monitored and controlled on anindividual basis. It is also within the scope of the invention to have aplurality of automated tissue engineering systems operating andnetworked under the control of a remote computer.

Although preferred embodiments of the invention have been describedherein in detail, it will be understood by those skilled in the art thatvariations may be made thereto without departing from the spirit of theinvention or the scope of the appended claims.

1-137. (canceled)
 138. An automated tissue engineering systemcomprising; a housing; at least one bioreactor supported by saidhousing, said bioreactor facilitating physiological cellular functionsand/ or the generation of one or more tissue constructs from cell and/ortissue sources; a fluid containment system supported by said housing andin fluid communication with said bioreactor, one or more sensorsassociated with one or more of said housing, bioreactor or fluidcontainment system for monitoring parameters related to saidphysiological cellular functions and/or generation of tissue constructs;and a microprocessor linked to one or more of said sensors.
 139. Thesystem of claim 138, wherein said bioreactor comprises a bioreactorhousing; one or more inlet ports and one or more outlet ports for mediaflow; and at least one chamber defined within said bioreactor housingfor receiving a variety of cells and/or tissues.
 140. The system ofclaim 139, wherein said bioreactor housing comprises a lid, said one ormore inlet ports and outlet ports being provided within said bioreactorhousing and/or said lid.
 141. The system of claim 138, wherein saidchamber is selected from the group consisting of a tissue digestionchamber, culture/proliferation chamber, differentiation/tissue formationchamber and combinations thereof.
 142. The system of claim 139, whereintwo or more chambers are provided operably connected within saidbioreactor.
 143. The system of claim 139, wherein said chamber housesone or more substrates and/or scaffolds.
 144. The system of claim 143,wherein said substrate is a contained suspension of micro-carrier. 145.The system of claims 139, wherein said chamber contains a plurality ofzones.
 146. The system of claim 145, wherein said plurality of zones mayeach accommodate a scaffold and/or substrate.
 147. The system of claim142, wherein at least one of said two or more chambers and saidbioreactors are one of at least independently operable andco-operatively operable.
 148. The system of claim 142, wherein at leastone of said chambers and bioreactors are operably connected to providefor the exchange of one or more of fluids, cells and tissues between atleast one of said chambers and said bioreactors.
 149. The system ofclaim 148, wherein at least one of said chambers and said bioreactor areconnected via at least one of a passageway, tubing, connector, valve,pump, filter, fluid access port, in-line gas exchange membrane, in-linesensor and void in a separation wall.
 150. The system of claim 138,wherein said bioreactor is removably accomodated with said fluidcontainment system via said inlet port and said outlet port.
 151. Anautomated tissue engineering system comprising; a housing; at least onetissue engineering module removably accommodated within said housing,said tissue engineering module comprising a support structure that holdsat least one bioreactor, said bioreactor facilitating at least one ofcell culture and tissue engineering functions, a fluid containmentsystem in fluid communication with said bioreactor, and one or moresensors for monitoring parameters related to at least one of said cellculture and tissue engineering functions; and a microprocessor disposedin said housing and linked to said tissue engineering module, saidmicroprocessor controlling the operation of said tissue engineeringmodule.
 152. The tissue engineering system of claim 151, wherein saidbioreactor comprises a bioreactor housing having one or more inlet portsand one or more outlet ports for media flow; and at least one chamberdefined within said bioreactor housing for receiving at least one ofsaid cells and tissues and facilitating said cell culture and tissueengineering functions.
 153. The tissue engineering system of claim 152,wherein said chamber houses one or more substrates and/or scaffolds.154. The tissue engineering system of claim 152, wherein at least oneof, said two or more chambers, and said bioreactors are at least one ofindependently operable and cooperatively operable.
 155. The tissueengineering system of claim 151, wherein at least one of said chambersand said bioreactors are operably connected to provide for the exchangeof at least one of said fluids, cells and tissues between at least oneof said chambers and said bioreactors.
 156. The tissue engineeringsystem of claim 153, wherein said chamber contains a plurality of zonesto contain a plurality of substrates and/ or scaffolds.
 157. The tissueengineering system of claim 151, wherein said fluid containment systemcomprises a plurality of flexible reservoirs connected by flexibletubing for supplying and retrieving fluid to and from said bioreactor.158. The tissue engineering system of claim 151, wherein said housingcomprises one or more environmental sensors and an environmental controlunit to maintain said environmental conditions within one or more ofsaid housing, tissue engineering module, bioreactor, fluid containmentsystem, and said flexible reservoirs.
 159. The tissue engineering systemof claim 151, wherein said housing has at least one insertion slot forinsertion of said tissue engineering module via at least one set ofguides for receiving said support structure.
 160. The tissue engineeringsystem of claim 159, wherein said insertion slot and said guides arevertically or horizontally orientated with respect to said housing. 161.The tissue engineering system of claim 159, wherein said insertion slothas a movable door and a locking mechanism.
 162. The tissue engineeringsystem of claim 151, wherein said system additionally comprises a userinterface in operation with said microprocessor, said user interfaceproviding for the entry of user inputs to said microprocessor and/or theoutput of system status.
 163. The tissue engineering system of claim162, further comprising a data system to permit input, output,recording, transfer and storage of information.
 164. The tissueengineering system of claim 163, further comprising a computer andcommunications link.
 165. The tissue engineering system of claim 151,wherein said microprocessor performs quality control assessments of saidcell culture and tissue engineering functions.
 166. The tissueengineering system of claim 151, wherein said tissue engineering moduleadditionally comprises one or more microprocessors that are in operableconnection with said microprocessor disposed in said housing.
 167. Aportable and sterilizable tissue engineering module, the modulecomprising; a structural support holding at least one bioreactor, saidbioreactor facilitating cell culture and tissue engineering functions; afluid containment system in fluid communication with said bioreactor;and one or more sensors for monitoring parameters related to said cellculture and tissue engineering functions.
 168. The tissue engineeringmodule of claim 167, wherein said bioreactor comprises: a bioreactorhousing having one or more inlet ports and one or more outlet ports formedia flow; and at least one chamber defined within said bioreactorhousing for receiving at least one of said cells and tissues andfacilitating said cell culture and tissue engineering functions. 169.The tissue engineering module of claim 168, wherein said chamber housesone or more substrates and/or scaffolds.
 170. The tissue engineeringmodule of claim 168, wherein two or more chambers are provided operablyconnected within said bioreactor.
 171. The tissue engineering module ofclaim 167, wherein two or more bioreactors are operably connected. 172.The tissue engineering module of claim 170, wherein at least one of saidtwo or more chambers, and said bioreactors are independently operableand/or co-operatively operable.
 173. The tissue engineering module ofclaim 172, wherein said module comprises a first bioreactor, a secondbioreactor and a third bioreactor, said first bioreactor containing atissue digestion chamber, said second bioreactor containing a culture/proliferation chamber, said third bioreactor containing adifferentiation/tissue formation chamber, and wherein said first, secondand third bioreactors are operatively connected.
 174. The tissueengineering module of claim 170, wherein said chambers and/orbioreactors are operably connected to provide for the exchange offluids, cells and/or tissues between said chambers and/or bioreactors.175. The tissue engineering module of claim 168, wherein said chambercontains a plurality of zones to contain a plurality of substratesand/or scaffolds.
 176. The tissue engineering module of claim 167,wherein said fluid containment system comprises a plurality of flexiblereservoirs connected by flexible tubing for supplying and retrievingfluid to and from said bioreactor.
 177. The tissue engineering module ofclaim 176, wherein at least one of said flexible reservoir and saidtubing is provided with fluid access ports for the loading or removal ofmaterial from said fluid containment system.
 178. The tissue engineeringmodule of claim 167, wherein a plurality of fluid flow control valvesare provided in operable connection with fluid containment system tocontrol the flow of fluid with said fluid containment system and saidbioreactor.
 179. The tissue engineering module of claim 178, whereinsaid module additionally comprises one or more pump units in connectionwith said fluid containment system for the pumping of fluid throughoutsaid fluid containment system.
 180. The tissue engineering module ofclaim 167, wherein a fluid flow plate is mounted or provided integral tosaid structural support, said fluid flow plate being operationallyconnected to said fluid control valves to direct fluid flow within andbetween said fluid containment system and said bioreactor.
 181. Thetissue engineering module of claim 167, wherein said tissue engineeringmodule additionally comprises a heating and mixing chamber to heat andmix fluids flowing to said bioreactor.
 182. The tissue engineeringmodule of claim 167, wherein one or more gas exchange membranes areprovided within one of or between said fluid containment system andbioreactor, said gas exchange membranes permitting the transfer ofgaseous products into or out of fluid flowing to or resident in saidbioreactor.
 183. The tissue engineering module of claim 167, whereinsaid module additionally comprises a thermoelectric element in operableconnection with said bioreactor.
 184. The tissue engineering module ofclaim 167, wherein said module additionally comprises one or moremicroprocessors in operable connection with said sensors.
 185. Thetissue engineering module of claim 167, wherein said module additionallycomprises one or more access ports, said access ports being operativelylinked with at least one of said bioreactor and said fluid containmentsystem, said access ports providing for sterile loading or removal ofcells, fluids, cell and tissue culture media, growth factors,pharmaceutical agents, quality control reagents, quality control samplesand other materials.
 186. The tissue engineering module of claim 185,wherein said access ports are operatively linked to a syringe manifoldintegrated with said structural support.
 187. The tissue engineeringmodule of claim 167, wherein said bioreactor is integrally mounted tosaid structural support.
 188. The tissue engineering module of claim167, wherein said bioreactor is detachable from said structural support.189. The tissue engineering module of claim 167, wherein a camera isprovided for visual inspection within said bioreactor.
 190. The tissueengineering module of claim 167, wherein said module additionallycomprises an identifying element.
 191. The tissue engineering module ofclaim 167, wherein said module is sterilizable before use and disposableafter use.
 192. A bioreactor for facilitating and supporting cellularfunctions and/or the generation of tissue constructs, said bioreactorcomprising; a bioreactor housing; one or more inlet ports and one ormore outlet ports for media flow; at least one chamber defined withinsaid bioreactor housing for facilitating and supporting cellularfunctions and/or the generation of one or more tissue constructs fromcell and/ or tissue sources; and one or more sensors for monitoringparameters related to said cellular functions and/or generation oftissue constructs within said at least one chamber.
 193. The bioreactorof claim 192, wherein said bioreactor housing comprises a lid, said lid.194. The bioreactor of claim 192, wherein said bioreactor comprises asingle culture/proliferation chamber having at least one of aculture/proliferation scaffold and a substrate supported therein. 195.The bioreactor of claim 192, wherein said bioreactor comprises aculture/proliferation chamber connected to a downstreamdifferentiation/tissue formation chamber, said culture/proliferationchamber having at least one of a proliferation scaffold and a substratesupported therein, said differentiation/ tissue formation chamber havingan implantable differentiation scaffold supported therein.
 196. Thebioreactor of claim 192, wherein a connecting passageway is providedbetween said culture/proliferation chamber and saiddifferentiation/tissue formation chamber, said connecting passagewayfacilitating movement of cells released from said proliferation scaffoldand/or substrate to said differentiation scaffold.
 197. The bioreactorof claim 196, further comprising a digestion chamber for the digestionof tissue biopsy material, said digestion chamber being upstream of saidculture/proliferation chamber.
 198. The bioreactor of claim 195, whereinone or more filters are provided at a location selected from upstream ofsaid proliferation scaffold, upstream of said differentiation scaffold,upstream to said outlet port and combinations thereof.
 199. Thebioreactor of claim 196, wherein said differentiation/tissue formationchamber contains a plurality of zones to contain a plurality ofdifferentiation scaffolds and/ or substrates.
 200. The bioreactor ofclaim 192, wherein said bioreactor has a sampling port that isoperatively connected to one or more of said chambers.
 201. Thebioreactor of claim 192, wherein a gas exchange membrane forms part ofsaid chamber.
 202. The bioreactor of claim 192, wherein said bioreactoris removably accomodated with a fluid containment system via said inletport and said outlet port.
 203. The bioreactor of claim 192, wherein amixing diaphragm forms part of said chamber and is operably connected toat least one of a mixing actuator and a mixing drive.
 204. Thebioreactor of claim 203, wherein said mixing diaphragm is incorporatedwithin said bioreactor.
 205. The bioreactor of claim 192, wherein saidbioreactor is operatively connected to at least one of an impactactuator and an impact drive.
 206. The bioreactor of claim 192, whereinsaid chamber further supports physiological stimulation of residentcells and/or tissues.
 207. The bioreactor of claim 192, furthercomprising a micro-loading diaphragm in operable connection with atleast one of a micro-loading actuator and a micro-loading drive. 208.The bioreactor of claim 206, wherein said stimulation is performed bysupplying an electric field.
 209. The bioreactor of claim 192, whereinsaid bioreactor is operationally accommodated within a tissueengineering module comprising a fluid containment system affixed to astructural support and in fluid communication with said bioreactor, andwherein said fluid containment system comprises a plurality of flexiblereservoirs connected by flexible tubing for supplying and retrievingfluid to and from said bioreactor.
 210. The bioreactor of claim 209,wherein said tissue engineering module is accommodated within a housingof an automated tissue engineering system, said system comprising amicroprocessor for operation with said module and wherein saidmicroprocessor controls the functioning of said module.
 211. Thebioreactor of claim 192, wherein said bioreactor additionally comprisesan optical probe.
 212. The bioreactor of claim 192, wherein saidbioreactor is sterilizable before use and disposable after use.
 213. Amethod for the automated digestion of a tissue biopsy, the methodcomprising; loading a tissue biopsy within a bioreactor connected with amedia reservoir and flow system, said bioreactor having one or moresensors to detect physiological conditions within said bioreactor forassessment by a microprocessor; providing tissue digestion enzymeswithin said bioreactor; and monitoring and maintaining digestionconditions within said bioreactor for a sufficient period of time for adesired level of tissue digestion.
 214. A method for the automatedproliferation of cells, said method comprising; seeding cells onto aproliferation substrate or scaffold supported within a bioreactorconnected with a media reservoir and flow system, said bioreactor havingone or more sensors to detect physiological conditions within saidbioreactor for assessment by a microprocessor; and monitoring andmaintaining suitable culturing conditions within said bioreactor for asufficient period of time for a desired level of cell proliferation.215. A method for the automated differentiation of cells, said methodcomprising; seeding cells onto a differentiation substrate or scaffoldsupported within a bioreactor connected with a media reservoir and flowsystem, said bioreactor having one or more sensors to detectphysiological conditions within said bioreactor for assessment by amicroprocessor; and monitoring and maintaining suitable culturingconditions within said bioreactor for a sufficient period of time for adesired level of cell differentiation.
 216. A method for the productionof a tissue construct, said method comprising; seeding cells onto ascaffold supported within a bioreactor, said bioreactor connected with amedia reservoir and flow system, said bioreactor having one or moresensors to detect physiological conditions within said bioreactor to amicroprocessor, and monitoring and maintaining suitable culturingconditions within said bioreactor for a sufficient period of time forsaid cells to express extracellular matrix that provides structuralsupport for the tissue construct.
 217. An automated method for digestinga tissue biopsy to provide primary cells, including precursor cells, andfurther proliferating and differentiating the cells to enable theformation of a tissue implant, the method comprising; loading a tissuebiopsy within a bioreactor connected with a media reservoir and flowsystem, said bioreactor having one or more sensors to detectphysiological conditions within said bioreactor for assessment by amicroprocessor; providing tissue digestion enzymes; monitoring andmaintaining suitable digestion conditions within said bioreactor for asufficient period of time to obtain disassociated cells; seeding thedisassociated cells onto a proliferation substrate or scaffold supportedwithin a bioreactor connected with a media reservoir and flow system,said bioreactor having one or, more sensors to detect physiologicalconditions within said bioreactor for assessment by a microprocessor,monitoring and maintaining suitable culturing conditions within saidbioreactor for a sufficient period of time to obtain the desired levelof cell proliferation and expansion; releasing the expanded cells fromthe proliferation substrate or scaffold; seeding the expanded cells ontoa differentiation substrate or scaffold supported within a bioreactorconnected with a media reservoir and flow system, said bioreactor havingone or more sensors to detect physiological conditions within saidbioreactor for assessment by a microprocessor, and monitoring andmaintaining suitable culturing conditions within said bioreactor for asufficient period of time to obtain a tissue implant.
 218. A method forproviding a skeletal implant, the method comprising; seeding osteogenicand/ or osteoprogenitor cells onto a porous scaffold of a bonebiomaterial supported within a bioreactor connected with a mediareservoir and flow system, said bioreactor having one or more sensors todetect physiological conditions within said bioreactor for assessment bya microprocessor; and monitoring and maintaining suitable conditionswithin said bioreactor for a sufficient period of time to allow theosteogenic and/ or osteoprogenitor cells to proliferate and/ordifferentiate throughout the scaffold to provide a tissue implant fororthopedic applications.
 219. A method for providing a cartilageimplant, the method comprising; seeding chondrogenic and/orchondroprogenitor cells onto a porous scaffold of a biomaterialsupported within a bioreactor connected with a media reservoir and flowsystem, said bioreactor having one or more sensors to detectphysiological conditions within said bioreactor for assessment by amicroprocessor; and monitoring and maintaining suitable conditionswithin said bioreactor for a sufficient period of time to allow thechondrogenic and/or chondroprogenitor cells to proliferate and/ordifferentiate throughout the scaffold to provide a cartilage implant.220. A method for providing an implant for reestablishing the innernucleus of a spinal disc, the method comprising; seeding nucleuspulposus cells within a scaffold a porous scaffold of a biomaterialsupported within a bioreactor connected with a media reservoir and flowsystem, said bioreactor having one or more sensors to detectphysiological conditions within said bioreactor to a microprocessor; andmonitoring and maintaining suitable conditions within said bioreactorfor a sufficient period of time to allow proliferation and/ordifferentiation of the nucleus pulposus cells and the expression ofextracellular matrix components characteristic of the nucleus pulposus.221. A method for washing cells, said method comprising: loading a cellsuspension containing one or more undesired chemicals into a chamber,continuously recirculating the cell suspension from the chamber througha cross-flow filtration module that comprises a membrane impermeable tosaid cells but permeable to said undesired chemicals to provide a washedcell suspension; and collecting the washed cell suspension.
 222. Amethod for enrichment of cells, said method comprising: loading a cellsuspension containing excessive cell suspension volume into a chamber;continuously recirculating the cell suspension from the chamber througha cross-flow filtration module that comprises a membrane impermeable tothe cells but allowing the excessive cell suspension volume to beremoved and collected.